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入 札 公 告次のとおり一般競争入札に付します。令和６年４月２２日国立研究開発法人国立環境研究所理事長 木本 昌秀１．競争入札に付する事項(１)件 名：【電子入札システム対応】令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務(２)契約期間：契約締結日から令和７年２月２８日まで(３)仕 様：仕様書による。(４)履行場所：仕様書による。２．競争参加資格(１)令和４・５・６年度環境省競争参加資格(全省庁統一資格)の「役務の提供等」の「調査・研究」において、「Ｂ」、「Ｃ」又は「Ｄ」の等級に格付けされている者であること。(２)国立研究開発法人国立環境研究所契約事務取扱細則第５条の規定に該当しない者であること。なお、未成年者、被保佐人又は被補助人であって、契約締結のために必要な同意を得ている者については、同条中、特別の理由がある場合に該当する。(３)国立研究開発法人国立環境研究所契約事務取扱細則第６条の規定に該当しない者であること。(４)契約者等から取引停止の措置を受けている期間中の者でないこと。(５)入札説明書において示す暴力団排除等に関する誓約事項に誓約できる者であること。３．電子入札システムの利用本件調達は電子入札システムで行う。なお、同システムによりがたい者は紙入札方式によることができる。・https://www.ebs-cloud.fwd.ne.jp/CALS/Accepter/index.jsp?name1=06A0064006A00600４．入札説明書等の交付場所(１)入札の方法等は別途交付する入札説明書によるので、必ず参照すること。(２)交付場所〒３０５－８５０６ 茨城県つくば市小野川１６－２国立研究開発法人国立環境研究所 総務部会計課契約第一係及び当研究所ＨＰ上ＴＥＬ ０２９－８５０－２７７５ ＦＡＸ ０２９－８５０－２３８８(担当：濱田)５．入札説明書等に対する質問令和６年４月３０日(火)１７時００分までに電子メールにより送付すること(送付先:chotatsu@nies.go.jp)。なお、メール送信後、前記４(２)に電話し、受信を確認すること。また、メールの件名は【質問の提出(令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務)(担当：濱田)】とすること。６．回答書閲覧期間及び場所令和６年５月８日(水)１０時００分から令和６年５月２２日(水)１１時００分まで当研究所ＨＰ上(本ページ(※))において閲覧可能である。ただし、質問のない場合は掲示しない。※https://www.nies.go.jp/osirase/chotatsu/kokoku/index.html７．入札参加資格証明書類の提出期限(１)提出期限及び提出場所令和６年５月１５日(水)１６時００分まで ４．(２)に示すとおり(２)提出方法書面の持参又は郵送(書留郵便等の配達記録が残る方法に限り、受領期間必着とする)によるものとする。なお、電子入札システム(同システムにより入札する者に限る。)若しくは電子メール(送付先：chotatsu@nies.go.jp)による電子データの提出も可とする。ただし、電子入札システムまたは電子メールによるデータ送信後、前記４．(２)に電話し、受信を確認すること。また、電子メールの場合、件名は【入札参加資格証明書類の提出(令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務)(担当：濱田)】とすること。８．入札及び開札の日時及び場所令和６年５月２２日(水)１１時００分国立研究開発法人国立環境研究所 研究本館Ⅱ １階 第１会議室(茨城県つくば市小野川１６－２)９．入札方法入札金額については、１．(１)の業務に関する一切の費用を含めた額とする。落札決定に当たっては、入札書に記載された金額に課税対象金額の１０％に相当する額を加算した金額(当該金額に１円未満の端数があるときは、その金額を切り捨てるものとする。)をもって落札価格とするので、入札参加者は、消費税及び地方消費税に係る課税事業者であるか免税事業者であるかを問わず見積もった契約金額から課税額を除いた金額を入札書に記載すること。１０．その他留意事項(１)入札及び契約手続きにおいて使用する言語及び通貨は、日本語及び日本国通貨に限る。(２)入札保証金 免除(３)契約保証金 免除(４)入札の無効 本公告に示した競争参加資格のない者がした入札及び入札に関する条件に違反した入札書は無効とする。(５)契約書作成の要否 要(６)落札者の決定方法入札書に記載されている入札書の提出方法、競争参加資格、仕様等の要求要件を全て満たし、仕様書において明らかにした要求要件のうち必須とされた項目の最低限の要求要件を全て満たし、当該入札書の入札価格が国立研究開発法人国立環境研究所契約事務取扱細則第１３条の規定に基づいて作成された予定価格の制限の範囲内で最低価格をもって有効な入札を行った入札者を落札者とする。

入 札 説 明 書【電子入札システム対応】令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務令和６年４月国立研究開発法人国立環境研究所当研究所の一般競争に係る入札公告(令和６年４月２２日付)に基づく入札については、関係法令に定めるもののほか、この入札説明書による。１．競争入札に付する事項(１)件 名 【電子入札システム対応】令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務(２)契約期間 契約締結日から令和７年２月２８日まで(３)仕 様 仕様書による。(４)履行場所 仕様書による。(５)入札保証金 免除(６)契約保証金 免除２．競争参加に必要な資格(１)令和４・５・６年度環境省競争参加資格(全省庁統一資格)の「役務の提供等」の「調査・研究」において、「Ｂ」、「Ｃ」又は「Ｄ」の等級に格付けされている者であること。(２)国立研究開発法人国立環境研究所契約事務取扱細則第５条の規定に該当しない者であること。なお、未成年者、被保佐人又は被補助人であって、契約締結のために必要な同意を得ている者については、同条中、特別の理由がある場合に該当する。(３)国立研究開発法人国立環境研究所契約事務取扱細則第６条の規定に該当しない者であること。(４)契約者等から取引停止の措置を受けている期間中の者でないこと。(５)入札説明書において示す暴力団排除等に関する誓約事項に誓約できる者であること。３．入札心得(１)入札参加者は、仕様書及び添付書類を熟読のうえ、入札しなければならない。(２)入札参加者は、前項の書類について疑義があるときは、関係職員の説明を求めることができる。(３)入札参加者は、入札後、仕様書及び添付書類についての不明等を理由として異議を申し立てることはできない。４．電子入札システムの利用本件調達は電子入札システムで行うため、同システムの電子認証(代表者又はその委任を受けた者のＩＣカードに限る。)を取得していること。・https://www.ebs-cloud.fwd.ne.jp/CALS/Accepter/index.jsp?name1=06A0064006A00600なお、同システムによりがたい者は、紙入札方式によることができる。ただし、紙入札方式参加届(別紙１)を７．(１)①に示す期限までに提出すること。提出は、書面の持参若しくは郵送又は電子メールによること。５．入札及び開札の日時及び場所令和６年５月２２日(水)１１時００分国立研究開発法人国立環境研究所 研究本館Ⅱ １階 第１会議室(茨城県つくば市小野川１６－２)６．入札説明書等に対する質問(１) 入札説明書(契約書案(別紙５)含む)に対する質問がある場合においては、次に従い、質問書(指定様式(※))を提出すること。※当研究所ＷＥＢサイトに掲載(本公告掲載先と同一ページ)①提出期間：令和６年４月２２日(月)から令和６年４月３０日(火)１７時００分まで。②提出方法：電子メールによる送付とする(送付先:chotatsu@nies.go.jp)。なお、メール送信後、後記１８．の担当あて電話し、受信を確認すること。また、電子メールの件名は【質問の提出(令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務)(担当：濱田)】とすること。(２)(１)の質問に対する回答書は、次のとおり閲覧に供する。①期 間：令和６年５月８日(水)１０時００分から令和６年５月２２日(水)１１時００分まで。②閲覧場所：当研究所ＷＥＢサイト(本公告掲載先と同一ページ)(３)(１)の質問がない場合、(２)については行わないものとする。７．入札参加資格証明書類の提出(１)入札に参加しようとする者は、本入札説明書２．(１)の証明書類を次に従い提出すること。※提出された書類については返却しない。①提出期限：令和６年５月１５日(水)１６時００分②提出方法：書面の持参又は郵送(書留郵便等の配達記録が残る方法に限り、受領期間必着とする)によるものとする。なお、電子入札システム(同システムにより入札する者に限る。)若しくは電子メール(送付先：chotatsu@nies.go.jp)による電子データの提出も可とする。ただし、電子入札システムまたは電子メールによるデータ送信後、後記１８．の担当あて電話し、受信を確認すること。また、電子メールの場合、件名は【入札参加資格証明書類の提出(令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務)(担当：濱田)】とすること。(２)(１)のとおり提出された書類による本競争参加の可否については、次の期間までに連絡をする。・入札日及び開札の２営業日前１６時００分８．入札及び開札(１)電子入札の場合①７．(１)①の日時までに、電子入札システムの証明書等提出画面において、２．(１)の競争参加資格を有することを証明する書類を提出すること。②５．の日時までに、同システムに定める手続に従って入札を行うこと。通信状況によっては当該期限内に入札情報が到着しない場合があるので、時間的余裕を持って行うこと。③入札金額については、１．(１)の業務に関する一切の費用を含めた額とする。④落札決定に当たっては、入札書に記載された金額に課税対象金額の１０％に相当する額を加算した金額(当該金額に１円未満の端数があるときは、その金額を切り捨てるものとする。)をもって落札価格とするので、入札参加者は、消費税及び地方消費税に係る課税事業者であるか免税事業者であるかを問わず見積もった契約金額から課税額を除いた金額を入札書に入力すること。⑤同システムにより入札した場合には、本入札説明書において示す暴力団排除等に関する誓約事項に誓約したものとして取り扱うこととする。⑥入札者又は代理人等は、開札時刻に同システムの端末の前で待機しなければならない。⑦事由のいかんにかかわらず入札の引換え、変更又は取消しを行うことができない。⑧入札参加者が連合し、又は不穏の行動をなす等の場合において、入札を公正に執行することができないと認められるときは、当該入札参加者を入札に参加させず、又は入札の執行を延期し、若しくは取りやめることがある。(２)紙入札の場合①入札書(別紙２)には、入札参加者の住所、氏名を記入し、金額の記入はアラビア数字を用いて鮮明に記載すること。また、郵送による提出の際は入札書に入札回数(第○回)を記載すること。②入札書及び入札に係る文書に使用する言語は、日本語に限るものとし、また入札金額は、日本国通貨による表示に限るものとする。③入札金額については、１．(１)の業務に関する一切の費用を含めた額とする。④落札決定に当たっては、入札書に記載された金額に課税対象金額の１０％に相当する額を加算した金額(当該金額に１円未満の端数があるときは、その金額を切り捨てるものとする。

)をもって落札価格とするので、入札参加者は、消費税及び地方消費税に係る課税事業者であるか免税事業者であるかを問わず見積もった契約金額から課税額を除いた金額を入札書に記載すること。⑤入札書は、別紙の書式により作成し、封かんの上で持参又は郵送により提出するものとする。⑥入札書を持参する場合は、入札書を封かんし、入札参加者の商号又は名称、入札件名及び開札日時を記載し、入札及び開札日に入札箱に投入すること。⑦新型コロナウイルスによる感染症(ＣＯＶＩＤ－１９)の感染拡大防止のため、当面の間郵送による入札書の提出は３通まで認めることとする。入札書を郵送により提出する場合は、二重封筒とし、表封筒に入札書在中の旨を朱書し、中封筒に入札参加者の入札参加者の商号又は名称、入札件名及び開札日時並びに入札回数(○回目)を記載して書留郵便(配達証明付)により、次に従い郵送すること。提出期限：入札及び開札の前日(※)１６時００分※土・日曜日、祝祭日及び年末年始(１２月２９日から１月３日)を除く。⑧入札参加者は、入札書を提出する際には、本入札説明書２．(１)の競争参加資格を有することを証明する書類を提出すること。⑨入札参加者は、代理人又は復代理人(以下「代理人等」という。)をして入札させるときは、その委任状(別紙３、４)を持参させなければならない。⑩入札参加者又はその代理人等は、当該入札に対する他の入札参加者の代理をすることができない。⑪開札は、入札参加者の面前で行う。ただし、入札参加者又はその代理人等が開札場所に出席しないときは、入札執行事務に関係のない職員を立会させて開札する。この場合、異議の申し立てはできない。⑫入札参加者又はその代理人等は、開札時刻後においては、開札場に入場することはできない。⑬提出済の入札書は、その事由のいかんにかかわらず引換え、変更又は取消しを行うことができない。⑭入札参加者が連合し、又は不穏の行動をなす等の場合において、入札を公正に執行することができないと認められるときは、当該入札参加者を入札に参加させず、又は入札の執行を延期し、若しくは取りやめることがある。９．入札の無効次の各号に該当する入札書は無効とする。(１)競争に参加する資格を有しない者の提出した入札書(２)紙入札において、委任状を持参しない代理人等の提出した入札書(３)紙入札において、記名を欠いた入札書(４)入札金額の記載が不明確な入札書(５)入札金額の記載を訂正した入札書(６)誤字、脱字等により意志表示が不明瞭である入札書(７)明らかに連合によると認められる入札書(８)同一事項の入札について、他の入札参加者の代理人等を兼ねた者の入札書(９)同一入札執行回について、入札参加者又はその代理人等が二通以上の入札書を提出した場合(10)その他の入札に関する条件に違反した入札書１０．落札の決定本入札説明書２．の競争参加資格及び仕様書等の要求要件を全て満たし、当該入札書の入札価格が国立研究開発法人国立環境研究所契約事務取扱細則第１３条の規定に基づいて作成された予定価格の範囲内で、最低の価格をもって有効な入札を行った者を落札者とする。１１．再度入札開札した場合において、入札参加者の入札のうち予定価格の制限に達した価格の入札が無いときは、直ちに再度の入札を行う。なお、再度入札の回数は原則として２回を限度とする。ただし、郵便による入札を行い、開札当日に入札参加者又はその代理人等が開札場所に出席しない場合においては、入札書の提出数以降の再度入札による入札に参加できないため注意すること。１２．同価格の入札が２人以上ある場合の落札者の決定(１)落札者となるべき同価格の入札をした者が２人以上あるときは、電子入札システムによる電子くじにより落札者を決定する。電子入札システムにより入札を行う場合は、入札時に任意の３桁の数字を入力すること。紙入札による場合は、入札書(別紙２)の記載欄に任意の３桁の数字を記載すること。なお、入力された数字は乱数処理により変換された数字により落札者を決定するため、指定した数字が直接判定に用いられるものではない。(２)前項の場合において、数字の指定を行わない者があるときは、職員が任意の数字を入力する。１３．落札内訳書の提出(１)落札者は、落札者の決定後すみやかに落札額に応じた内訳書を提出すること。なお、内訳書は可能な限り詳細に記載すること。(２)内訳書の様式は自由とする。(３)内訳書は返却しない。１４．契約書等の提出(１)落札者は、契約書案(別紙５)により、契約書を作成及び記名押印の上、速やかにこれを契約担当者等に提出しなければならない。なお、当該契約書案の記載内容に質問等がある場合は、前記６．により質問書を提出すること。(２)契約書及び契約に係る文書に使用する言語及び通貨は、日本語及び日本国通貨による。(３)契約担当者等が契約の相手方とともに契約書に記名押印しなければ、本契約は確定しないものとする。１５．契約者の氏名国立研究開発法人国立環境研究所 理事長 木本 昌秀１６．入札結果及び契約情報の公表について① 入札結果の公表落札者が決定したときは、その入札結果(落札者を含めた入札者全員の商号又は名称及び入札価格)について、開札場において発表するとともに電子入札システムにおいて公表する予定である。② 契約情報の公表契約を締結したときは、後日当該契約情報を当法人のＨＰにおいて公表する。独立行政法人が行う契約については、「独立行政法人の事務・事業の見直しの基本方針(平成２２年１２月７日閣議決定)」において、独立行政法人と一定の関係を有する法人と契約をする場合には、当該法人への再就職の状況、当該法人との間の取引等の状況について、情報を公開する等の取組を進めることとされている。これに基づき、以下のとおり、当法人との関係に係る情報を当法人のＨＰで公表することとするので、所要の情報の当法人への提供及び情報の公表に同意の上で、応札若しくは応募又は契約の締結を行っていただくようお願いする。なお、応札若しくは応募又は契約の締結をもって、同意されたものとみなすこととする。

1) 公表の対象となる契約先次のいずれにも該当する契約先ア．当法人において役員を経験した者が再就職をしていること又は課長相当職以上の職を経験した者が役員、顧問等として再就職していることイ．当法人との間の取引高が、総売上高又は事業収入の３分の１以上を占めていること2) 公表する情報上記に該当する契約先との契約(予定価格が一定の金額を超えない契約や光熱水料の支出に係る契約等は対象外)について、契約ごとに、物品・役務等の名称及び数量、契約締結日、契約先の名称、契約金額等と併せ、次に掲げる情報を公表する。ア．前記②1)アに該当する再就職者の人数、職名及び当法人における最終職名イ．当法人との間の取引高ウ．総売上高又は事業収入に占める当法人との間の取引高の割合が、次の区分のいずれかに該当する旨・３分の１以上２分の１未満・２分の１以上３分の２未満・３分の２以上エ．一者応札又は一者応募である場合はその旨3) 提供を求める情報ア．契約締結時点における前記②1)アに該当する再就職者に係る情報(人数、職名及び当法人における最終職名)イ．直近の事業年度における総売上高又は事業収入及び当法人との間の取引高4) 公表の時期契約締結日の翌日から起算して原則７２日以内(４月中に締結した契約については原則９３日以内)１７．電子入札システムの操作及び障害発生時の問合せ先電子入札システム ポータルサイトアドレスhttps://www.nies.go.jp/osirase/chotatsu/kokoku/e-bidding/index.htmlヘルプデスク 0570-021-777(受付時間：平日9:00～12:00及び13:00～17:30)Email:sys-e-cydeenasphelp.rx@ml.hitachi-systems.com１８．提出書類送付先(担当部署)〒３０５－８５０６茨城県つくば市小野川１６－２国立研究開発法人国立環境研究所 総務部会計課契約第一係 濱田ＴＥＬ ０２９－８５０－２７７５E-mail chotatsu@nies.go.jp(別紙１)年 月 日紙入札方式参加届国立研究開発法人国立環境研究所理事長 殿住 所商号又は名称代表者名下記入札案件について、紙入札方式での参加をいたします。件名： 令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務担当者等連絡先部署名 ：担当者名：責任者名：ＴＥＬ ：E-mail ：(別紙２)入 札 書金 円電子くじに入力する数字(任意の３桁)：件名 令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務上記金額をもって貴所入札説明書承諾のうえ入札します。御採用のうえは確実に履行いたします。なお、入札説明書別紙６の暴力団排除等に関する誓約事項に誓約します。年 月 日住 所商号又は名称代 表 者 名国立研究開発法人国立環境研究所 理事長 殿担当者等連絡先部署名 ：担当者名：責任者名：ＴＥＬ ：E-mail ：＜記入例＞入 札 書金 円電子くじに入力する数字(任意の３桁)：件名 令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務上記金額をもって貴所入札説明書承諾のうえ入札します。御採用のうえは確実に履行いたします。なお、入札説明書別紙６の暴力団排除等に関する誓約事項に誓約します。××年××月××日住 所 ○○県○○市○○１－２－３商号又は名称 株 式 会 社 △ △ △ △代 表 者 名 代表取締役 □ □ □ □＜(復)代理人 ◎ ◎ ◎ ◎ ＞※代理人又は復代理人が入札する際は、代表者に代わり代理人又は復代理人が記名すること国立研究開発法人国立環境研究所 理事長 殿担当者等連絡先部署名 ：担当者名：責任者名：ＴＥＬ ：E-mail ：(別紙３)年 月 日委 任 状国立研究開発法人国立環境研究所 理事長 殿住 所商号又は名称代 表 者 名今般、私は、 を代理人と定め、令和６年４月２２日付け公示された国立研究開発法人国立環境研究所の「令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務」に関し、下記の権限を委任いたします。受任者：住 所商号又は名称役職・氏名記1.本入札に係る一切の権限2.1.の事項に係る復代理人を選任すること担当者等連絡先部署名 ：担当者名：責任者名：ＴＥＬ ：E-mail ：(別紙４)年 月 日委 任 状国立研究開発法人国立環境研究所 理事長 殿住 所商号又は名称氏 名今般、私は、 を復代理人と定め、令和６年４月２２日付け公示された国立研究開発法人国立環境研究所の「令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務」に関し、下記の権限を委任いたします。受任者：住 所商号又は名称役職・氏名記1.本入札に係る一切の権限担当者等連絡先部署名 ：担当者名：責任者名：ＴＥＬ ：E-mail ：(別紙５)契 約 書(案)国立研究開発法人国立環境研究所 理事長 木本 昌秀(以下「甲」という。)と、(以下「乙」という。)とは、次の条項により契約を締結する。１．件 名 令和６年度ヨコエビを用いた慢性底質試験法の検証に係る業務２．契約金額 金 円(うち消費税及び地方消費税額 円)３．契約期間 自 契約締結日 至 令和７年２月２８日４．契約保証金 免除５．契約履行の場所及び業務内容 別添仕様書のとおり(信義誠実の原則)第１条 甲乙両者は、信義を重んじ誠実に本契約を履行しなければならない。(権利義務の譲渡等)第２条 乙は、本契約によって生じる権利又は義務の全部若しくは一部を、甲の承諾を得た場合を除き第三者に譲渡し、又は承継させてはならない。ただし、信用保証協会及び中小企業信用保険法施行令(昭和２５年政令第３５０号)第１条の３に規定する金融機関に対して売掛債権を譲渡する場合にあっては、この限りでない。(義務の履行)第３条 乙は、別添仕様書に基づき、頭書の金額をもって頭書の期間中に義務を完全に履行しなければならない。(再委託等の禁止)第４条 乙は、業務の処理を第三者(再委託等先が乙の子会社(会社法(平成１７年法律第８６号)第２条第３号に規定する子会社をいう。)である場合も含む。以下同じ。)に委託し又は請け負わせてはならない。但し、再委託等承認申請書(別紙)を甲に提出し、甲の承認を得たときは、この限りではない。(監督職員)第５条 甲は、乙の業務実施について、自己に代って監督又は指示する監督職員を選定することができる。２ 監督職員は、本契約書及び仕様書に定められた事項の範囲内において業務の施行に立会い、又は必要な指示を与えることができる。※再委託等の取り扱いについては、仕様書及び「契約における再委託等の取扱いについて」(当研究所ＨＰに掲載)を参照すること。掲載先：https://www.nies.go.jp/osirase/chotatsu/saiitaku.pdf(業務の報告等)第６条 甲は、必要と認めたときは、乙に対して業務の実施状況について報告を受け、又は説明を求める等の措置をとることができる。２ 乙は、甲が前項の報告を依頼し、又は書類の提出を求めたときはすみやかにこれに応じるものとする。

(業務内容の変更)第７条 甲は、必要がある場合には、業務の内容を変更することができる。この場合において、契約金額又は契約期間を変更するときは、甲乙協議して書面によりこれを定めるものとする。(契約の解除)第８条 甲は、次の各号の一に該当するときは、催告することなくこの契約の全部又は一部を解除することができる。一 乙の責に帰する事由により、乙がこの契約の全部又は一部を履行する見込みがないと認められるとき。二 乙が第４条、第１７条又は第１８条の規定に違反したとき。三 乙又はその使用人が甲の行う監督及び検査に際し不正行為を行い、又は監督者等の職務の執行を妨げたとき。四 履行期限内に成果品の提出がなかったとき。２ 甲は、乙が次の各号の一に該当すると認められるときは、催告することなくこの契約を解除することができる。一 法人等(個人、法人又は団体をいう。)の役員等(個人である場合はその者、法人である場合は役員又は支店若しくは営業所(常時契約を締結する事務所をいう。)の代表者、団体である場合は代表者、理事等、その他経営に実質的に関与している者をいう。)が、暴力団(暴力団員による不当な行為の防止等に関する法律(平成３年法律第７７号)第２条第２号に規定する暴力団をいう。以下同じ)又は暴力団員(同法第２条第６号に規定する暴力団員をいう。以下同じ。)であるとき二 役員等が、自己、自社若しくは第三者の不正の利益を図る目的、又は第三者に損害を加える目的をもって、暴力団又は暴力団員を利用するなどしているとき三 役員等が、暴力団又は暴力団員に対して、資金等を供給し、又は便宜を供与するなど直接的あるいは積極的に暴力団の維持、運営に協力し、若しくは関与しているとき四 役員等が、暴力団又は暴力団員であることを知りながらこれを不当に利用するなどしているとき五 役員等が、暴力団又は暴力団員と社会的に非難されるべき関係を有しているとき３ 甲は、乙が自ら又は第三者を利用して次の各号の一に該当する行為をした場合は、催告することなくこの契約を解除することができる。一 暴力的な要求行為二 法的な責任を超えた不当な要求行為三 取引に関して脅迫的な言動をし、又は暴力を用いる行為四 偽計又は威力を用いて甲等の業務を妨害する行為五 その他前各号に準ずる行為４ 甲は、前三項の規定により、この契約の全部又は一部を解除した場合は、既に乙に支払った契約金額の全部又は一部を乙に返還させることができる。(再受任者等に関する契約解除)第９条 乙は、契約後に再受任者等(再受任者、及び乙又は再受任者が当該契約に関して個別に契約する場合の当該契約の相手方をいう。以下同じ。)が第８条第２項及び第３項の一に該当する者(以下「解除対象者」という。)であることが判明したときは、直ちに当該再受任者等との契約を解除し、又は再受任者等に対し契約を解除させるようにしなければならない。２ 甲は、乙が再受任者等が解除対象者であることを知りながら契約し、若しくは再受任者等の契約を承認したとき、又は正当な理由がないのに前項の規定に反して当該再受任者等との契約を解除せず、若しくは再受任者等に対し契約を解除させるための措置を講じないときは、催告することなくこの契約を解除することができる。(違約金)第 10 条 次に掲げる場合のいずれかに該当したときは、乙は、甲の請求に基づき、契約金額の１００分の１０に相当する金額を違約金として甲の指定する期間内に支払わなければならない。一 甲が第８条又は第９条第２項の規定により契約の全部又は一部を解除したとき。二 乙について破産手続開始の決定があった場合において、破産法(平成１６年法律第７５号)の規定により選任された破産管財人が契約を解除したとき。三 乙について更生手続開始の決定があった場合において、会社更生法(平成１４年法律第１５４号)の規定により選任された管財人が契約を解除したとき。四 乙について再生手続開始の決定があった場合において、民事再生法(平成１１年法律第２２５号)の規定により選任された再生債務者等が契約を解除したとき。五 この契約に関し、乙が私的独占の禁止及び公正取引の確保に関する法律(昭和２２年法律第５４号。以下「独占禁止法」という。)第３条の規定に違反し、又は乙が構成事業者である事業者団体が独占禁止法第８条第１号の規定に違反したことにより、公正取引委員会が乙に対し、独占禁止法第７条の２第１項(独占禁止法第８条の３において準用する場合を含む。)の規定に基づく課徴金の納付命令(以下「納付命令」という。)を行い、当該納付命令が確定したとき(確定した当該納付命令が独占禁止法第６３条第２項の規定により取り消された場合を含む。)。六 この契約に関し、乙が独占禁止法第３条の規定に違反し、又は乙が構成事業者である事業者団体が独占禁止法第８条第１号の規定に違反したことにより、公正取引委員会が乙又は当該事業者団体(以下「乙等」という。)に対し、独占禁止法第７条若しくは第８条の２の規定に基づく排除措置命令(以下「排除措置命令」という。)を行い、当該排除措置命令が確定したとき。七 この契約以外の乙の取引行為に関して、乙が独占禁止法第３条の規定に違反し、又は乙が構成事業者である事業者団体が独占禁止法第８条第１号の規定に違反したことにより、公正取引委員会が、乙等に対し、納付命令又は排除措置命令を行い、これらの命令が確定した場合において、これらの命令に乙等に独占禁止法第３条又は第８条第１号の規定に違反する行為があったとされた期間及び当該違反する行為の対象となった取引分野が示され、この契約が、当該期間(これらの命令に係る事件について、公正取引委員会が乙に対し納付命令を行い、これが確定したときは、当該納付命令における課徴金の計算の基礎である当該違反する行為の実行期間を除く。)に入札(見積書の提出を含む。)が行われたものであり、かつ、当該取引分野に該当するものであるとき。八 この契約に関し、乙(法人にあっては、その役員又は使用人を含む。)の刑法(明治４０年法律第４５号)第９６条の６又は独占禁止法第８９条第１項若しくは第９５条第１項第１号に規定する刑が確定したとき。２ 前項の規定は、甲に生じた実際の損害の額が違約金の額を超える場合において、甲がその超える分の損害を損害金として請求することを妨げない。(報告)第11条 乙は、作業終了後すみやかに甲に作業終了の報告をしなければならない。

(検査)第12条 甲は、前条の報告があったときは、当該届出を受理した日から１０日以内に検査を行わなければならない。(契約金の支払)第13条 乙は、前条の検査に合格したときは、甲に契約金の支払を請求するものとする。２ 甲は、前項の規定により、乙から適法な契約金の請求を受けたときは、請求書を受理した日から６０日以内に支払うものとする。(損害賠償)第14条 甲は、第８条又は第９条第２項の規定によりこの契約を解除した場合は、これにより乙に生じた損害について、何ら賠償ないし補償することは要しない。(担保責任)第 15 条 甲は、乙が本契約履行後に提出した成果品について１年以内に契約の内容に適合しないものであることを発見したときは、契約不適合である旨を乙に通知し、修補又は既に支払った契約金額の一部を返還させることができるものとする。(延滞金)第 16 条 乙は、第８条第４項の規定による契約金額の返還又は第１０条の規定による違約金等の支払いを甲の指定する期間内に行わないときは、当該期間を経過した日から支払いをする日までの日数に応じ、民法(明治２９年法律第８９号)第４０４条で定める法定利率で計算した額の延滞金を甲に支払わなければならない。(守秘義務)第 17 条 甲及び乙は、この契約の履行に際し、知り得た相手方の秘密を第三者に漏らし、又は利用してはならない。(個人情報の取扱い)第 18 条 乙は、甲から預託を受けた個人情報(生存する個人に関する情報であって、当該情報に含まれる氏名、生年月日その他の記述又は個人別に付された番号、記号その他の符号により当該個人を識別できるもの(当該情報のみでは識別できないが、他の情報と容易に照会することができ、それにより当該個人を識別できるものを含む。)をいう。以下同じ。)について、善良な管理者の注意をもって取扱う義務を負わなければならない。２ 乙は次の各号に掲げる行為をしてはならない。ただし、事前に甲の承認を受けた場合は、この限りではない。(１)甲から預託を受けた個人情報を第三者(再委託等する場合における再委託等先を含む。)に預託若しくは提供又はその内容を知らせること。(２)甲から預託を受けた個人情報を本契約の目的の範囲を超えて使用、複製、又は改変すること。３ 乙は、甲から預託を受けた個人情報の漏洩、滅失、毀損の防止その他の個人情報の適切な管理のために必要な措置を講じなければならない。４ 乙は、甲から預託を受けた個人情報について、作業終了、又は解除をした後に速やかに甲にその媒体を返還するとともに、乙が保存している当該個人情報について、復元不可能な状態に消去し、その旨を甲に通知しなければならない。ただし、甲が別に指示したときは、その指示によるものとする。５ 乙は、預託を受けた個人情報の取扱いに係る業務を第三者に再委託等してはならない。

ただし、上層水及び間隙水濃度は請負者にて適切な前処理の後に測定する。試験期間中における分析対象の試料数は 60 個とする。また、ヨコエビ底質試験の実施に当たって手順の改訂が必要であると考えられる事項を抽出する。６ 成果物の提出請負者は、試験が終了次第、可能な限り 1 か月以内に業務結果の速報をNIES担当者へ提出するものとする。その後、業務結果報告書の草案を作成し、NIES 担当者と協議の上、業務契約期間終了時までに報告書の電子ファイルを電子メール等によって送付するものとする。７ 著作権等の扱い(1)請負者は、本業務の目的として作成される成果物に関し著作権法第 27条及び第28条を含む著作権の全てをNIESに無償で譲渡するものとする。(2)請負者は、成果物に関する著作者人格権(著作権法第18条から第20条までに規定された権利をいう。)を行使しないものとする。ただし、NIES が承認した場合は、この限りではない。(3)上記(1)及び(2)にかかわらず、成果物に請負者が既に著作権を保有しているもの(以下「既存著作物」という。)が組み込まれている場合は、当該既存著作物の著作権についてのみ、請負者に帰属する。提出される成果物に第三者が権利を有する著作物が含まれる場合には、請負者が当該著作物の使用に必要な費用の負担及び使用許諾契約等に係る一切の手続を行うものとする。８ 情報セキュリティの確保請負者は、国立研究開発法人国立環境研究所情報セキュリティポリシーを遵守し、情報セキュリティを確保するものとする。特に下記の点に留意すること。なお、国立研究開発法人国立環境研究所情報セキュリティポリシーは以下URL において公開している。(https://www.nies.go.jp/security/sec_policy.pdf)①請負者は、請負業務の開始時に、請負業務に係る情報セキュリティ対策の遵守方法及び管理体制、事故時における緊急時の連絡体制について、NIES 担当者に書面で提出すること。②請負者は、NIESから要機密情報を提供された場合には、当該情報の機密性の格付けに応じて適切に取り扱われるための措置を講ずること。③請負者は、国立研究開発法人国立環境研究所セキュリティポリシーの履行が不十分と見なされるとき又は請負者において請負業務に係る情報セキュリティ事故が発生したときは、必要に応じてNIESの行う情報セキュリティ監査を受け入れること。④請負者は、NIESから提供された要機密情報が業務終了等により不要になった場合には、確実に返却し又は廃棄し、文書にて報告すること。⑤業務に用いる電算機(パソコン等)は、使用者の履歴が残るものを用いてこれを保存するとともに、施錠など適切な盗難防止の措置を講じること。また、Winny 等の P2P ソフトをインストールしていないことが確認できたもののみを使用すること。⑥再委託することとなる場合は、事前の承諾を得て再委託先にも以上と同様の制限を課して契約すること。９ 検 査本業務終了後、NIES 担当者による本仕様書に基づく検査に合格しなければならない。１０ 協議事項本業務に関し疑義等を生じたときは、速やかにNIES 担当者と協議の上、その指示に従うものとする。１１ そ の 他請負者は、本業務実施に係る活動において、国等による環境物品等の調達の推進等に関する法律(グリーン購入法)を推進するよう努めるとともに、物品の納入等に際しては、基本方針で定められた自動車を利用するよう努めるものとする。1OECD/OCDEOECD GUIDELINE FOR TESTING OF CHEMICALSSediment-water Amphipod Toxicity Test Using Spiked SedimentPart I: 10-day sub-chronic toxicity testPart II: 42-day chronic toxicity testPART I: 10-DAY SUB-CHRONIC TOXICITY TESTINTRODUCTION1. Benthic animals are subject to potentially high exposure to chemicals present in sedimentenvironments and should therefore be given preferential attention. Among these benthicorganisms, amphipods play an important role in sediment ecosystems. This Test Guideline isdesigned to assess the effects of exposure of chemicals to the freshwater benthic amphipodHyalella azteca and other well documented freshwater amphipods (e.g., Gammarus pulex,Gammarus pseudolimnaeus, and Gammarus fossarum). The description about other freshwateramphipods may be deleted, if no laboratory uses these species in the inter-laboratory ring testIt is based on existing toxicity test protocols for H. azteca which have been developed by UnitedStates Environmental Protect Agency (USEPA, 2000; USEPA 2016a; ASTM, 2020) andEnvironment and Climate Change Canada (2017)2. The exposure scenario used in this guideline is spiking of sediment with the test chemicalThe scenario of spiking sediment is intended to simulate accumulated levels of chemicalspersisting in the sediment3. Chemicals that need to be tested towards benthic organisms usually persist in thiscompartment over long time periods. Benthic organisms may be exposed via several routesThe relative importance of each exposure route, and the time taken for each to contribute to theoverall toxic effects, is dependent on the physical-chemical properties of the chemicalconcerned among others. For strongly adsorbing chemicals (e.g., with log KOW > 5) or forchemicals covalently binding to sediment, ingestion of contaminated food may be a significantexposure route. In order not to underestimate the toxicity of highly lipophilic chemicals, the useof food added to the sediment before application of the test chemical may be considered (seeparagraph 36)別添24. The measured endpoints for 10-day sub-chronic toxicity test (Part I) are survival and growth.

The measured endpoints for the 42-day chronic test (Part II) are survival, growth, andreproduction.

5. The use of formulated sediment (also called reconstituted, artificial, or synthetic sediment)is recommended. Formulated sediment has several advantages over natural sediment (alsocalled field-collected or environmental sediment):✓ the experimental variability is reduced because it forms a reproducible "standardizedmatrix." and the need to find uncontaminated and clean sediment sources is eliminated;✓ the tests can be initiated at any time without encountering seasonal variability in the testsediment and there is no need to pre-treat the sediment to remove indigenous fauna;✓ the use of formulated sediment also reduces the cost associated with the field collection ofsufficient amounts of sediment for routine testing.

6. However, natural sediments will be considered acceptable if it can be demonstrated that thesediment does not have concentrations of contaminants which reduced survival or growthof test organisms and the amphipods in the control meet the validity criteria.

7. Definitions are given in ANNEX 1.

PRINCIPLE OF THE TEST8. Seven to eight days old juvenile amphipods are exposed to a concentration range of the testchemical in sediment - water systems. The test chemical is spiked into the sediment and thespiked sediment is transferred to test beakers after sediment conditioning is completed.

Juveniles are subsequently introduced into test beakers. Survival and dry weight (and bodylength as an option) are measured at the end of the 10-day test. These data are analyzed eitherby using a regression model in order to estimate the concentration that would cause x%reduction in survival or dry weight (e.g., EC50 etc.), or by using statistical hypothesis testingto determine a NOEC/LOEC. The latter requires comparison of effect values with controlvalues using statistical tests.

INITIAL CONSIDERATIONS9. It is recommended to know information about chemical-specific properties such as thestructural formula, molecular weight, purity, stability in water and light, acid dissociationconstant (pKa), organic carbon partition coefficient (KOC) and n-octanol water partitioncoefficient (KOW), water solubility, and vapor pressure, as well as results of a test for readybiodegradability OECD TG 301 (OECD, 1992) or OECD TG 310 (OECD, 2006a). Conduct ofthis test guideline without the information listed above should be carefully considered as the3study design will be dependent on the physical-chemical properties of the test chemical andcould lead to meaningless or difficult to interpret results.

10. A validated analytical method, of known accuracy, precision, and sensitivity, for thequantification of the test chemical and potential degradation and transformation products in thetest solution and sediment should be available (OECD, 2014), where technically feasible.

Performance parameters should be reported (e.g., accuracy, precision, limit of detection, limitof quantification, specificity, working range).

11. If the Test Guideline is used for the testing of a mixture, a substance of Unknown orVariable composition, Complex reaction products or Biological materials (UVCB) or a multi-constituent substance, its composition should, as far as possible, be characterized, e.g., by thechemical identity of its constituents, their quantitative occurrence and their chemical-specificproperties. Recommendations about the testing of difficult test chemicals like mixtures, UVCBsor multi-constituent substances are given in Guidance Document No. 23 (OECD, 2019).

VALIDITY OF THE TEST12. For the test to be valid the following conditions apply:✓ The mean mortality in the controls should not exceed 20% at the end of the test;✓ If the mean dry weight at the start of the test is 0.02 mg per individual or more, the meandry weight in the control at the end of the test should be at least 2.5 times larger than themean initial dry weight. If the mean initial dry weight is less than 0.02 mg, the mean dryweight in the control at the end should be 0.05 mg per individual or more; This criterionshould be revised if other freshwater amphipods are included in this guideline.

✓ Throughout the test period, the oxygen concentration in overlying water should be greaterthan 2.5 mg/L, and the pH of overlying water should be between 6 and 9;✓ Throughout the test period, the water temperature should be within the range of 23 ± 3°C,and the mean water temperature should be within the range of 23 ± 1°C. This criterionshould be revised if other freshwater amphipods are included in this guideline.

DESCRIPTION OF THE METHODTest vessels13. Normal laboratory equipment for the conduct of this assay, with appropriate documentationto validate that the equipment is working correctly, include:✓ oxygen meter4✓ pH meter✓ light meter✓ adequate apparatus for temperature control✓ equipment for determination of hardness and ammonia concentration of water✓ equipment of determination of total organic carbon content✓ equipment for the determination of concentration of test chemical in test solution andsedimentTest vessels14. The test is conducted preferably in glass 300 ml beakers that are 6 to 8 cm in diameter.

Other vessels are suitable, but they should guarantee a suitable depth of overlying water andsediment (at a minimum of 1.5 cm in depth of sediment). The ratio of the depth of the sedimentlayer to the depth of the overlying water should be within the range of from 1:1.75 (e.g., 100mL sediment and 175 mL water) to 1:4 (e.g., 55 mL sediment and 220 mL water). Test vesselsand other apparatus that will come into contact with the test system should be made entirely ofglass or other chemically inert material (e.g., polytetrafluoroethylene [PTFE]). Test vesselsmust be cleaned thoroughly before and after use.

Selection of species15. The preferred species for this test is Hyalella azteca. Details of culture methods are givenin ANNEX 2. H. azteca is known as a complex of many cryptic species; therefore the strainand genetic information should be confirmed before testing (Major et al. 2013) but are notrequired prior to every test if organisms come from an in-house culture. Other well documentedfreshwater species may also be used, for example Gammarus pulex, Gammaruspseudolimnaeus, and Gammarus fossarum (USEPA, 2016b). The sentence about otherfreshwater amphipods may be deleted if no laboratory uses these species in the inter-laboratoryring test.

Sediment16. Formulated sediment should preferably be used. However, if natural sediment is used, itshould be characterized (at least pH, organic carbon content; in addition, it is recommended todetermine other parameters determination of other parameters such as C/N ratio andgranulometry are also recommended), and it should not have concentrations of contaminantswhich reduced survival or growth of test organisms. Also, natural sediment should not containother organisms that might compete with or consume the amphipods. The following formulatedsediment, according to the OECD Guideline 218 (OECD, 2004), is recommended for use in5this test: It is also recommended that, before it is used in an amphipod toxicity test, the sedimentbe conditioned for seven days under the same conditions which prevail in the subsequent test(see paragraph 27).

a. 4-5% (dry weight) peat (e.g., Sphagnum): a close to pH 5.5 to 6.0 as possible; it isimportant to use peat in powder form, finely ground (particle size ≤ 1mm) and only airdried.

b. 20% (dry weight) kaolin clay (kaolinite content preferably above 30%).

c. 75-76% (dry weight) quartz sand (fine sand should be predominate with more than 50%of the particles between 50 and 200 μm).

d. Deionized water is added to obtain a moisture content of the final mixture in a range of30-50%.

e. Calcium carbonate of chemically pure quality (CaCO3) is added to adjust the pH of thefinal mixture of the sediment to 7.0 ± 0.5. Organic carbon content of the final mixtureshould be 2% (± 0.5%) and is to be adjusted by the use of appropriate amounts of peatand sand, according to (a) and (c).

17. The source of peat, kaolin clay, and sand should be known. The sediment components willbe analyzed to identify background contaminants such as heavy metals, organochlorinecompounds, and organophosphorus compounds. An example for the preparation of theformulated sediment is described in ANNEX 3. Mixing of dry constituents is also acceptable ifit is demonstrated that after addition of overlying water a separation of sediment constituents(e.g., floating of peat particles) does not occur, and that the peat or the sediment is sufficientlyconditioned.

18. Other types of formulated sediment have been proposed for H. azteca and can be used. Forexample, Environment and Climate Change Canada (2017) and ISO (2013) recommend themixture of sand, aluminium (III) oxide, iron (III) oxide, peat, and clay as formulated sediment.

Kemble et al. (1999) proposed another type of formulated sediment consisting of sand, silt, clay,dolomite, α-cellulose, and humic acid. Note that cellulose may show lower KOC values for a testchemical than peat moss and natural sediment (Cui and Gan, 2013), thereby increasing in thebioavailability and toxicity of the chemical.

19. Sediment type and sediment properties can substantially affect the fate and bioavailabilityof the test chemical, and therefore uptake by the test organism and subsequent toxicity. If naturalsediment is used, it is necessary to report sediment characteristics that may have an influenceon bioavailability and therefore toxicity. Total organic carbon usually provides the main bindingphase for many types of chemicals and therefore its concentration in natural sediment shouldbe measured so that normalisation of toxicity data to organic carbon can be done (ECHA 2017a,6b).

Water20. Reconstituted water (ANNEX 2) should preferably be used. However, any suitable water,natural water (surface or ground water), or dechlorinated tap water are acceptable as culturingwater and test water if amphipods will survive in it for the duration of the culturing and testingwithout showing signs of stress. At the start of the test, the pH of the test water should bebetween 6 and 9. For H. azteca, test water should exceed both 0.02 mg Br/L and 15 mg Cl/L(Ivey and Ingersoll, 2016) and 90 mg/L water hardness as CaCO3, although a concentrationhigher than 0.04 mg Br/L is recommended (ASTM, 2020). If bromide and chlorideconcentrations are below these levels, NaBr and NaCl should be added to test water. The sametype of water should be used throughout the whole study. The water quality characteristics listedin ANNEX 2 should be measured at least twice a year, if natural or tap water is used or when itis suspected that these characteristics may have changed significantly. If using natural water,special care should be taken considering the characteristics mentioned.

Stock solutions - spiked sediments21. Spiked sediments of the chosen concentration are usually prepared by addition of a solutionof the test chemical directly to the sediment. A stock solution of the test chemical dissolved indeionized water is mixed with the formulated sediment by rolling mill, feed mixer or handmixing. If poorly soluble in water, the test chemical can be dissolved in as small a volume aspossible of a suitable organic solvent (e.g., hexane, acetone, methanol, ethanol, or other volatilesolvents). This solution is then mixed with small aliquots of fine quartz sand (e.g., 10 to 100 g).

The solvent is allowed to evaporate, and it has to be totally removed from sand; the sand is thenmixed with the suitable amount of sediment per test concentration or per test beaker. Onlyagents which volatilize readily can be used to solubilize, disperse or emulsify the test chemical.

It should be borne in mind that the sand provided by the test chemical and sand mixture, has tobe taken into account when preparing the sediment (i.e., the sediment should thus be preparedwith less sand). Care should be taken to ensure that the test chemical added to sediment isthoroughly and evenly distributed within the sediment. It is recommended that subsamplesshould be analyzed to determine degree of homogeneity.

TEST DESIGN22. The test design relates to the selection of the number and spacing of the test concentrations,the number of vessels at each concentration. Designs for EC point estimation, for estimation ofNOEC, and for conducting a limit test are described.

Design for analysis by regression723. The effect concentration (e.g., EC50) and the concentration range, over which the effect ofthe test chemical is of interest, should be spanned by the concentrations included in the test.

Generally, the accuracy and especially the validity, with which estimates of effectconcentrations (ECx) can be made, is improved when the effect concentration is within therange of concentrations tested. Extrapolation outside the concentration range tested should beavoided. A preliminary range-finding test is helpful for selecting the range of concentrations tobe used.

24. If the ECx is to be estimated in 10-day sub-chronic test, at least five concentrations and atleast four replicates for each concentration should be tested. In any case, it is advisable thatsufficient test concentrations are used to allow good model estimation. The spacing factorbetween concentrations should not be greater than two (an exception could be made in caseswhen the dose response curve has a shallow slope). The number of replicates at each treatmentcan be reduced if the number of test concentrations with different responses is increased.

Increasing the number of replicates or reducing the size of the test concentration intervals tendsto lead to narrower confidence intervals for the test. In the 42-day chronic test, more replicatesfor each concentration are required (see Part II).

Design for estimation of a NOEC/LOEC25. If the NOEC or LOEC are to be estimated, five test concentrations with at least fourreplicates (but preferably 8 or more) should be used and the spacing factor betweenconcentrations should not be greater than two. The number of replicates should be sufficient toensure adequate statistical power to detect a 20% difference from the control at the 5% level ofsignificance (p = 0.05) and depends on the variability of the data. For example, if five replicatesare used per treatment, the coefficient of variation (CV) needs to be 10% or lower to detect a20% difference from the control with a power of 90% (ASTM, 2020). Limit test26. A limit test may be performed (one test concentration and control) if no effects were seenin the preliminary range-finding test. The purpose of the limit test is to perform a test at aconcentration sufficiently high to enable decision makers to exclude possible toxic effects oftest chemical, and the limit is set at a concentration which is not expected to appear in anysituation. A sediment concentration of 1000 mg/kg (dry weight) is recommended. Usually, atleast five replicates for both the treatment and control are necessary. Adequate statistical powerto detect a 20% difference from the control at the 5 % level of significance (p = 0.05) should bedemonstrated. With metric response (body weight), the t-test is a suitable statistical method ifdata meet the requirements of this test (normality, homogeneous variances). The unequal-variance t-test or a non-parametric test, such as the Wilcoxon-Mann-Whitney test may be used,if these requirements are not fulfilled.

8PROCEDUREConditions of exposurePreparation of spiked sediment -water system27. Sufficient time should be allowed after spiking for the spiked chemical to equilibrate withsediment components (USEPA 2000). Appropriate conditioning time is sediment and chemicalspecific. Organic chemicals with log KOW 6 can take 2 months or more (ASTM 2020). Metals may reach equilibriumwithin weeks to months, but the times differ depending on metals and sediments (ASTM 2020).

Periodic monitoring of chemical concentrations in pore water during sediment conditioning canbe useful as a means to assess the equilibration of the spiked sediments. The spiked sedimentshould be placed in a suitable and sealed container, and stored in the dark at 4°C to avoiddegradation of a test chemical before use.

28. The spiked sediment is transferred to the test beakers after conditioning periods andoverlying water is added to produce a sediment-water volume ratio of 1:1.75 to 1:4. The depthof the sediment layer should be more than 1.5 cm. To avoid separation of sediment ingredientsand re-suspension of fine material during addition of test water in the water column, thesediment can be covered with a disc (e.g., stainless steel or PTFE) while water is poured ontoit, and the disc removed immediately afterwards. Other devices may also be appropriate. Theaddition of sediment and overlying water is recommended to be performed at least 48 h beforeadding test organisms to allow for stabilization in test beakers (see paragraph 34).

Addition of test organisms29. Age of amphipods at the start of the test must be between 6- to 10-days old. To obtain lessvariable growth results among tests, wider age range is not allowed. Test organisms arecollected by isolating adult organisms for 24–48 h, then retrieving the young produced duringthat period. The collected young amphipods are 0–2 days old at the time of collection and willbe 6–10 days old after being held for 6–8 days. The age range within the population oforganisms used to start the test must be no more than 48 h (i.e., the range of 6- to 8-days old,7- to 9-days old, and 8- to 10-days old can be used).

30. Test organisms should be handled as little as possible. Ten amphipods should be introducedinto the overlying water below water-air surface in each beaker. The weight or size of the testorganisms at the start of exposure should be measured using 20 or more amphipods from thesame population used in the test to assess the growth at the end of the test. A relatively largenumber of amphipods (i.e., 20 or more) are needed for accurate weight measurement, becausedry weight of amphipod at 7–8 days old is as small as 0.020–0.035 mg per individual (ASTM,92020). It is recommended that the weight (and length) of amphipods at 6–10 days old shouldbe compared with the historical data of the laboratory.

Test concentrations31. A range-finding test may be helpful to determine the range of concentrations for thedefinitive test. For this purpose, a series of widely spaced concentrations of the test chemicalare used (e.g., a factor of 10). The amphipods are exposed to each concentration of the testchemical for a period which allows estimation of appropriate test concentrations, and noreplicates are required. The amphipods density per vessel should be the same as for thedefinitive test.

32. The test concentrations for the definitive test are decided based on the result of the range-finding test. At least five concentrations should be used and selected as described in paragraphs23 to 25.

Controls33. Control treatment without any test chemical but including sediment should be included inthe test with the appropriate number of replicates (see paragraphs 23-25). If a solvent is usedfor application of test chemical (see paragraph 21), the solvent control should be prepared byadding the solvent to sediment and additional control without solvent is not necessary.

Water renewal34. Either static, semi-static, or flow-through exposure system can be used. If static exposureis performed, overlying water should be aerated to provide oxygen. The aeration speed shouldbe slow (e.g., about 1 to 2 bubbles per second) not to re-suspend sediment particles. If overlyingwater is renewed during the test, renewal can be accomplished manually or automatically at therate of 2 volumes additions of overlying water per day. Automated water-delivery systems havebeen proposed for amphipod sediment toxicity tests (Zumwalt et al., 1994). The water-deliverysystem should be calibrated before a test is started to verify that the system is functioningproperly. If overlying water is manually renewed, water renewal should be done as early andlate in the day to minimize the length of time the system goes without renewal. Whether static,semi-static or flow-through exposure is performed, it is recommended to add the sediment tobeakers and start water renewal (if performed) at least 48 h before the addition of test organismsto allow for the transfer of the test chemical from the sediment to aqueous phase. Thisstabilization period is sediment and chemical specific, and can be in the order of hours and inrare cases extended up to several weeks (i.e., 4 to 5 weeks).

35. Although chemical concentrations are reduced or fluctuated in the overlying water due toperiodical or continuous water-renewal, organisms in direct contact with sediment generallyreceive a substantial proportion of a chemical dose directly from either the whole sediment or10from the pore water. This results in consistent toxicity levels among different exposure systems(i.e., static, semi-static, or flow-through) (Ankley et al., 1993; Hiki et al., 2023). However, sinceamphipods may avoid sediment and swim in overlying water (see paragraph 37), it isrecommended that chemical concentrations in overlying water should be measured as well asin pore water and sediment (paragraph 40).

Food36. Without addition of food, the test organisms may starve during exposures. It is necessary tofeed the amphipods, preferably daily or at least three times per week. As long as the validitycriteria are met, any type of food can be provided. However, the addition of excess or differenttypes of food should be avoided because it might alter the bioavailability of contaminants in thesediment and/or promote the growth of fungi or bacteria on the sediment surface. Here, twoexamples of feeding regime are shown. First, YCT (yeast, cerophyl, and trout chow) and flakefood (e.g., TetraMin or TetraFin) are provided as follows: 1.0 mL of YCT per beaker-daythroughout the 10-day in combination with 0.25 mg and 0.50 mg of flake food per beaker-dayfrom Day 0 to 6 and from Day 7 to 9, respectively. The other example of diet combination iscommercially available diatoms (Thalassiosira weissflogii) and flake food as follows (USEPA2016a; ASTM, 2020): 0.50 mg and 0.75 mg of diatom suspension from Day 0 to 6 and fromDay 7 to 9, respectively, and 0.25 mg and 0.50 mg of flake food per beaker-day from Day 0 to6 and Day 7 to 9, respectively. Any other diatoms or alga instead of Thalassiosira weissflogiimay also be successful, though their adequacy for use in testing should be verified. Note thateffects may be underestimated as food provided to the test organisms is not spiked with the testchemical. When testing strongly adsorbing chemicals (e.g., log KOW >5) or substancescovalently binding to sediment, food may be added to sediment before spiking of the testchemical and subsequent stabilization. For this, plant material should be used instead of fishfood, for example the addition of 0.5% (dry weight) finely ground leaves of stinging nettle(Urtica dioeca), mulberry (Morus alba), white clover (Trifolium repens), spinach (Spinaciaoleracea) or of other plant material (dried and milled wheat grass or alpha-cellulose) may beused (OECD, 2004). The applicability of plant materials to the amphipod sediment test remainsunclear, and their use should be verified during the development of this TG. Alternatively, field-collected sediment with sufficiently high nutritional content for the total test period could beused instead of standard OECD sediment which has lower nutritional value, as far as the validitycriteria are met.

Incubation conditions37. The test is conducted at a constant temperature of 23 ± 1°C. The water temperature shouldbe measured daily, and the average value over the test should be within the range of 23 ± 1°C.

The instantaneous temperature should be 23 ± 3°C. These sentences for temperature should be11revised if other freshwater amphipods are included in this guideline. Aeration can be providedthrough a glass Pasteur pipette fixed 2-3 cm above the sediment layer to maintain dissolvedoxygen in the overlying water above 2.5 mg/L (i.e., about 1 bubble/second in the overlyingwater). A 16 hours photoperiod is used and the light intensity should be 540 to 1080 lux.

ObservationsBehavior38. The test organism behavior (e.g., sediment avoidance) should be observed throughout thetest. Avoidance of amphipods from the sediment would result in a decreased exposure to thetest chemical, thereby reducing observed toxicity.

Mortality39. When the 10 day test is terminated, just before sieving the contents of a test vessel, all liveand apparently dead amphipods in water column or on the surface of the sediment should bepipetted. Then, the sediment from exposure beakers is sieved using a sieve (e.g., mesh size: 425µm) to retain the amphipods. A consistent amount of time should be taken to sieve contents ofeach test vessel and examine this closely for recovery of live or dead amphipods. Individualswhich are completely inactive but not obviously dead (e.g., not decomposing) should be heldin test water within a petri dish or other suitable container to assess the lethality closely using alow- power microscope or hand-held magnifying glass. Death is defined as the cessation of allvisible signs of movement or activity indicating life. Missing or unaccounted for amphipodsshould also be counted as dead, because dead amphipods are easily decomposed and missing.

Growth40. The dry weight of the surviving amphipod per test beaker is determined and the meanindividual dry weight per beaker calculated. Amphipods can be preserved in 8% sugar formalinsolution or other substitutes for formalin until the measurement. The sugar formalin solution isprepared adding 120 g of sucrose to 80 mL of formalin and mixed with a volume of 1 L usingdeionized water, then diluted with an equal volume of deionized water when used forpreservation. All fixed amphipods from a given test beaker are pooled together into a weigh panand dried for at least 24 hours at about 60 °C to a constant weight, and weighed to the nearest0.01 mg after being transferred in a desiccator at room temperature. Care should be taken tothoroughly rinse the sugar formalin from the preserved amphipods before weighing theseorganisms. Amphipod body length also can be measured as a growth endpoint from the base ofthe first antenna to the tip of the third uropod along the curve of the dorsal surface using animage processing software (e.g., Image J).

Analytical measurementConcentration of the test chemical in test beaker1241. For the analytical determination of the test chemical concentration, samples of the overlyingwater, the pore water and the sediment should be taken for analysis at least at the start and endof exposure, at least at the highest concentration and a lower one, but preferably at all testedconcentrations. These determinations of test chemical concentration inform about the behavior/partitioning of the tested chemical in the water-sediment system, and about the mass balancewithin the system (see ANNEX 5). 42. Centrifugation at, for example, 10,000 g and 4 °C for 30 min. is the recommended procedureto isolate pore water. However, if the test chemical is demonstrated not to adsorb to filters,filtration may also be acceptable. In some cases, it might not be possible to analyzeconcentrations in the pore water as the sample size is too small.

Physical-chemical parameters43. The pH and dissolved oxygen in overlying water should be measured at least in one vesselof each concentration at the start and end of the test. The temperature in overlying water shouldbe measured daily at least in one vessel of each concentration (see paragraph 37). Devices (e.g.,probes) used to measure pH, temperature, and dissolved oxygen should be inspected betweensamples to make sure that amphipods are not attached. Ammonia should be measured in thecontrols and one test vessel at the highest concentration at the start and the end of the test.

DATA AND REPORTINGTreatment and expression of results44. Effect concentrations should always be reported in terms of mg test chemical/kg drysediment. It is recommended to report mass balance calculations (see ANNEX 5) for gaininginformation on partitioning of the chemical in the various compartments. This is optionaldepending on the requirements in the respective regulations; it is also particularly important forchemicals that are difficult to test (concentrations poorly maintained in the test system). Theendpoints can be also expressed in mg chemical/L overlying water when deemed relevant.

45. To express the endpoints as nominal concentrations based on dry sediment, there should beevidence that the concentration of the chemical being tested has been satisfactorily maintainedin the sediment (i.e. between 80 % and 120 % of the nominal concentration throughout the test).

If the concentrations are expected to deviate by more than 20 %, all test concentrations shouldbe measured and more frequent analyses may be carried out.

For the sediment compartment, the endpoints should then be expressed in terms of geometricmean measured concentrations or in time-weighted arithmetic mean measured concentrationswhen the samples were taken in two or more unequal time intervals (with calculationsconsidering also the additional intermediate analytical measurements) in accordance withrecommendations in ANNEX 2 of OECD Guidance Document 23 (OECD, 2019) and examplesin ANNEX 6 of OECD TG211 (OECD, 2012). 13For the overlying water, the endpoints should be expressed similarly in terms of geometric meanmeasured concentrations or time-weighted arithmetic mean measured concentrations.

46. To convert concentration data expressed e.g. as mg/kg dry sediment into mg/kg organiccarbon (OC), the dry sediment concentration should be divided by the percent TOC (expressedas decimal), as follows:   ⁄ =    ⁄     ⁄47. To compute a point estimate for the ECx or LCx values, the per-vessel statistics may beused as true replicates. In calculating a confidence interval for any ECx, the variability amongvessels should be taken into account, or it should be shown that this variability is so small thatit can be ignored. When the model is fitted by Least Squares, a transformation should be appliedto the per-vessel statistics in order to improve the homogeneity of variance. However, ECxvalues should be calculated after the response is transformed back to the original value. Furtherinformation on statistics are given in OECD Document 54 on the Current Approaches in theStatistical Analysis of Ecotoxicity Data: A Guidance to Application (OECD, 2006b).

TEST REPORT48. The test report should include the following information:Test chemical:✓ physical nature and, where relevant, physical-chemical properties (water solubility, vapourpressure, partition coefficient in soil or in sediment, stability in water, etc);✓ chemical identification data (common name, chemical name, structural formula, CASnumber, etc.) including purity and analytical method for quantification of test chemical.

Test species:✓ test animals used: species, scientific name, source of organisms and breeding conditions;✓ information on handling of juvenile amphipods until the start of test;✓ age of test animals when added to test vessels.

Test conditions:✓ sediment used (i.e., formulated or natural sediment);✓ for natural sediment, location and description of sediment sampling site, including, ifpossible, contamination history; characteristics: pH, organic carbon content, C/N ratio andgranulometry (if appropriate). ✓ preparation of formulated sediment: ingredients and characteristics (organic carbon content,pH, moisture, etc. at the start of the test);✓ preparation of the test water (if reconstituted water is used) and characteristics (oxygen14concentration, pH, conductivity, hardness, etc.);✓ depth of sediment and overlying water;✓ volume of overlying and pore water; weight of wet sediment with and without pore water;✓ test vessels (material and size);✓ method of spiking sediment: test concentrations used, number of replicates and use ofsolvent if any;✓ stabilization phase of the spiked sediment-water system: duration and conditions,justification of extended stabilization (if appropriate);✓ water-renewal procedure used (i.e., static, semi-static, or flow-through) and frequency ofrenewal;✓ incubation conditions: temperature, light cycle and intensity, aeration (if provided);✓ detailed information on feeding including type of food, preparation, amount and feedingregime.

Results:✓ the nominal test concentrations, the measured test concentrations and the results of allanalyses to determine the concentration of the test chemical in the test vessel, includingassessment of mass balance if appropriate;✓ water quality within the test vessels, i.e. pH, temperature, dissolved oxygen, hardness andammonia;✓ number of surviving amphipods per vessel and concentration at the end of test;✓ mean individual dry weight of amphipods per vessel and concentration, if appropriate;✓ observed abnormal behavior (e.g., sediment avoidance), if any;✓ estimates of toxic endpoints, for example ECx (and associated confidence intervals),NOEC and/or LOEC, and the statistical methods used for their determination;✓ discussion of the results, including any influence on the outcome of the test resulting fromdeviations from this Guideline.

15PART II: 42-DAY REPRODUCTION TEST (USING H. AZTECA)** Currently, only the differences from Part I are shown. **INTRODUCTION49. The 10-day test which only quantifies effects on survival and growth can evaluate highlevels of toxicity, but may not be able to evaluate hazard at medium levels or to identify adverseeffects specific to reproductive endpoints. The method described in Part II measures survival,growth, and reproduction of H. azteca in a 42-day test. The procedure of this part is based onexisting toxicity test protocols for H. azteca which have been developed by United StatesEnvironmental Protect Agency (USEPA, 2000), International Organization for Standardization(ISO, 2013), and Environment and Climate Change Canada (2017).

PRINCIPLE OF THE TEST50. Seven to eight days old juvenile amphipods are exposed to a concentration range of the testsubstance in sediment-water systems. The test chemical is spiked into the sediment andjuveniles are subsequently introduced into test beakers in which the sediment and waterconcentrations have been stabilized. Survival, growth, and reproduction are measured at theend of the 42-day test. On Day 28, amphipods are transferred to test beakers without sediment,thereby making the collection of newborns feasible. This test design might allow amphipods torecover from effects of sediment exposure during the holding period in clean water from Day28 to 42; however, amphipods are exposed to sediment during critical developmental stagesbefore release of the first brood (USEPA, 2000).

VALIDITY OF THE TEST51. For the test to be valid the following conditions apply:✓ The mean mortality in the controls should not exceed 20% at the end of the test (i.e., Day42);✓ The mean dry weight in the controls should be 0.50 mg or larger per individual survivingadult amphipod at the end of test (i.e., Day 42); This criterion should be revised if otherfreshwater amphipods are included in this guideline.

✓ The mean reproduction in the controls should be 6.0 young or more per surviving adultfemale at the end of test (i.e., Day 42);✓ Throughout the test period, the oxygen concentration in overlying water should be greaterthan 2.5 mg/L, and the pH in overlying water should be between 6.0 and 9.0;16✓ Throughout the test period, the water temperature should be within the range of 23 ± 3°C,and the mean water temperature should be within the range of 23 ± 1°C. This criterionshould be revised if other freshwater amphipods are included in this guideline.

DESCRIPTION OF THE METHODDesign for analysis by regression and estimation of a NOEC/LOEC52. If the ECx and NOEC/LOEC are to be estimated, at least five concentrations and eightreplicates for each concentration should be tested. The large number of replicates in the 42-daytest is due to the fact that reproduction is often more variable than growth (reproductiontypically has coefficient of variation (CV) values of >20% compared to growth CV values of<10%) (USEPA, 2000). It is advisable that sufficient test concentrations are used to allow goodmodel estimation. The spacing factor between concentrations should not be greater than two(an exception could be made in cases when the dose response curve has a shallow slope). Thenumber of replicates at each treatment can be reduced if the number of test concentrations withdifferent responses is increased. Increasing the number of replicates or reducing the size of thetest concentration intervals tends to lead to narrower confidence intervals for the test.

PROCEDUREConditions of exposure53. The procedure and conditions of 42-day test, including preparation of spiked-sediment,water renewal, and incubation conditions are identical to those used in the10-day test. Thedifferences are outlined in the following paragraphs (paragraphs 54–59).

Addition of test organisms54. Age of H. azteca at the start of the test must be between 7- to 8-day old. The 42-day testshould start with a narrower range in size or age of H. azteca (i.e., 1-day range in age) to reducepotential variability in growth at the end of tests, compared to 10-day test. Test organisms arecollected by isolating adult organisms for 24 h, then retrieving the young produced during thatperiod. The collected young amphipods are 0–1 days old at the time of collection and will be7–8 days old after being held for 7 days. The age range within the population of organisms usedto start the test must be no more than 24 h (i.e., the range of 7- to 8-days old can be used). Sincethe sexes of juvenile amphipods cannot be identified, randomly selected 10 amphipods aretransferred to each test beaker. Test concentrations55. The results of a 10-day test (Part I) are can be used to determine the concentration levels in42-day test. A preliminary range-finding 42-day test is also helpful to determine the range of17concentrations for the definitive 42-day test.

Transferring to a water-only beaker56. On Day 28, the sediment is sieved using a sieve (e.g., mesh size: 300 µm) and the survivingadult amphipods in each sediment beaker are placed in 300-mL water-only beakers containing150 to 275 mL of clean water and 5 mL of quartz sand. If young amphipods are found whensieving the sediment, the number of young amphipods must be recorded. Either static, semi-static, or flow-through system can be used for 14 days of reproduction monitoring period. Food57. Without addition of food, the test organisms may starve during exposures. It is necessary tofeed the amphipods, preferably daily or at least three times per week. As long as the validitycriteria are met, any type of food can be provided. However, the addition of excess or differenttypes of food should be avoided because it might alter the bioavailability of contaminants in thesediment and/or promote the growth of fungi or bacteria on the sediment surface. Here, twoexamples of feeding regime are shown. First, YCT and flake food (e.g., TetraMin or TetraFin)are provided as follows: 1.0 mL of YCT per beaker-day throughout the 42-day in combinationwith 0.25 mg (the first week), 0.50 mg (the second week), 1.0 mg (the third week), 1.5 mg (thefourth week), 2.0 mg (the fifth week), and 2.5 mg (the sixth week) of flake food per beaker-day.

The other example of diet combination is commercially available diatoms (e.g., Thalassiosiraweissflogii) and flake food as follows: 0.50 mg (the first week), 0.75 mg (the second week), 1.0mg (the third week), 1.5 mg (the fourth week), 2.0 mg (the fifth week), and 2.5 mg (the sixthweek) of diatom suspension, and flake food as described in the first option. Any other diatomsor alga instead of Thalassiosira weissflogii can be applied if their suitability has beendemonstrated. When testing strongly adsorbing substances (e.g., log KOW >5) or substancescovalently binding to sediment, food may be added to sediment before spiking of the testsubstance and subsequent stabilization. For this, plant material should be used instead of fishfood, for example the addition of 0.5% (dry weight) finely ground leaves of stinging nettle(Urtica dioeca), mulberry (Morus alba), white clover (Trifolium repens), spinach (Spinaciaoleracea) or of other plant material (Cerophyl or alpha-cellulose) may be used (OECD, 2004).

The applicability of plant materials to the amphipod sediment test remains unclear, and theiruse should be verified during the development of this TG.

Ending a test58. On Days 35 and 42, all young amphipods are pipetted from a test beaker and the number iscounted. All live and apparently dead adult amphipods are counted without being transferredfrom the beaker, and their sexes are identified. The adult males are judged by the presence ofan enlarged second gnathopod and all other adults are considered as females. Technicians mustbe trained and able to discriminate male and female amphipods. Images of individualamphipods can be taken to help the discrimination of sexes. The number of young per surviving18female is calculated by dividing the total number of young produced in a replicate by thenumber of adult female organisms surviving on Day 42 in that replicate.

Analytical measurementConcentration of the test substance in test beaker59. When intermediate measurements are made (for example on day 7) and if the analysis needslarge samples which cannot be taken from test vessels without influencing the test system,analytical determinations should be performed on samples from additional test vessels treatedin the same way (including the presence of test organisms) but not used for biologicalobservations.

DATA AND REPORTINGTEST REPORT60. The test report should include the following information:Test chemical:✓ physical nature and, where relevant, physical-chemical properties (water solubility, vapourpressure, partition coefficient in soil or in sediment, stability in water, etc);✓ chemical identification data (common name, chemical name, structural formula, CASnumber, etc.) including purity and analytical method for quantification of test chemical.

Test species:✓ test animals used: species, scientific name, source of organisms and breeding conditions;✓ information on handling of juvenile amphipods until the start of test;✓ age of test animals when added to test vessels.

Test conditions:✓ sediment used (i.e., formulated or natural sediment);✓ for natural sediment, location and description of sediment sampling site, including, ifpossible, contamination history; characteristics: pH, organic carbon content, C/N ratio andgranulometry (if appropriate). ✓ preparation of formulated sediment: ingredients and characteristics (organic carbon content,pH, moisture, etc. at the start of the test);✓ preparation of the test water (if reconstituted water is used) and characteristics (oxygenconcentration, pH, conductivity, hardness, etc.);✓ depth of sediment and overlying water;✓ volume of overlying and pore water; weight of wet sediment with and without pore water;✓ test vessels (material and size);✓ method of spiking sediment: test concentrations used, number of replicates and use ofsolvent if any;19✓ stabilization phase of the spiked sediment-water system: duration and conditions,justification of extended stabilization (if appropriate);✓ water-renewal procedure used (i.e., static, semi-static, or flow-through) and frequency ofrenewal;✓ incubation conditions: temperature, light cycle and intensity, aeration (if provided);✓ detailed information on feeding including type of food, preparation, amount and feedingregime.

Results:✓ the nominal test concentrations, the measured test concentrations and the results of allanalyses to determine the concentration of the test chemical in the test vessel, includingassessment of mass balance if appropriate;✓ water quality within the test vessels, i.e. pH, temperature, dissolved oxygen, hardness andammonia;✓ number of surviving amphipods per vessel and concentration at the end of test;✓ mean individual dry weight of amphipods per vessel and concentration, if appropriate;✓ number of male and female adult amphipods per vessel and concentration at the end of test(i.e., Day 42);✓ number of young amphipods observed per vessel per day (i.e., Day 28, 35 and 42);✓ observed abnormal behavior (e.g., sediment avoidance), if any;✓ estimates of toxic endpoints, for example ECx (and associated confidence intervals),NOEC and/or LOEC, and the statistical methods used for their determination;✓ discussion of the results, including any influence on the outcome of the test resulting fromdeviations from this Guideline.

20REFERENCESAmerican Society for Testing and Materials (2020) Standard Test Method for Measuring theToxicity of Sediment- Associated Contaminants with Freshwater Invertebrates. E1706 – 20Ankley GT, Benoit DA, Hoke RA, Leonard EN, West CW, Phipps GL, Mattson VR, AndersonLA (1993) Development and evaluation of test methods for benthic invertebrates andsediments: Effects of flow rate and feeding on water quality and exposure conditions. Arch.

Environ. Contam. Toxicol. 25, 12–19.

Cui X, Gan J (2013) Comparing behavior of pyrethroids between formulated and naturalsediments. Environ Toxicol Chem 32(5): 1033-1039.

ECHA (2017a) Guidance on the Biocidal Regulation. Volume IV Environment – Assessmentand Evaluation (Parts B + C). Helsinki (FI).

ECHA (2017b) Guidance on information requirements and chemical safety assessment.

Chapter R.7b: endpoint specific guidance. Helsinki (FI).

Environment and Climate Change Canada (2017) Biological test method: test for survival andgrowth in sediment using the freshwater amphipod Hyalella azteca. Third Edition. RM.33.

September 2017.

Hiki K, Fischer CF, Nishimori T, Endo S, Watanabe H, Yamamoto H (2023) Influence of waterexchange rates on toxicity and bioaccumulation of hydrophobic organic chemicals in sedimenttoxicity tests. Environ. Sci. Processes Impacts 25 (3):609-620.

International Organization for Standardization (2013) Water Quality-Determination of Toxicityof Fresh Water Sediments Using Hyalella azteca. Report No. ISO 16303:2013 Geneva,Switzerland.

Ivey C, Ingersoll C (2016) Influence of bromide on the performance of the amphipod Hyalellaazteca in reconstituted waters. Environ Toxicol Chem 35(10): 2425-2429.

Kemble N, Dwyer C, Ingersoll C, Dawson T, Norberg-King T (1999) Tolerance of freshwatertest organisms to formulated sediments for use as control materials in whole-sediment toxicitytests. Environ Toxicol Chem 18(2): 222-230.

Major K, Soucek DJ, Giordano R, Wetzel MJ, Soto-Adames F (2013) The commonecotoxicology laboratory strain of Hyalella azteca is genetically distinct from most wild strainssampled in eastern North America. Environ Toxicol Chem 32(11): 2637-2647.

OECD (1992) Ready Biodegradability. Test Guideline 301.

21OECD (2004) Sediment-Water Chironomid Toxicity Using Spiked Sediment. Test Guideline218.

OECD (2006a) Ready Biodegradability – CO2 in sealed vessels (Headspace Test). TestGuideline 310.

OECD (2006b). Current approaches in the statistical analysis of ecotoxicity data: a guidance toapplication. Series on Testing and Assessment No. 54, OECD Paris.

OECD (2012) Daphnia magna Reproduction Test. Test Guideline 211.

OECD (2019). Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficulttest chemicals. OECD Environment, Health and Safety Publications, Series on Testing andAssessment No. 23 (second edition).

USEPA (2000) Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Second edition. EPA 600/R-99/064.

March 2000. Revision to the first edition dated June 1994.

USEPA (2016a) OCSPP 850.1735: Spiked Whole Sediment 10-Day Toxicity Test, FreshwaterInvertebrates. EPA 712/C/16/002. October 2016.

USEPA (2016b) OCSPP 850.1020: Gammarid Amphipod Acute Toxicity Test. EPA712/C/16/012. October 2016.

Zumwalt DC, Dwyer FJ, Greer IE, Ingersoll CG (1994) A water-renewal system that accuratelydelivers small volumes of water to exposure chambers. Environ Toxicol Chem 13(8): 1311–1314.

22ANNEX 1For the purpose of this guideline the following definitions are used:Conditioning period or aging period is the interval between spiking a test chemical and theaddition of the spiked sediment to test vessels.

Formulated sediment or reconstituted, artificial or synthetic sediment, is a mixture ofmaterials used to mimic the physical components of a natural sediment.

Overlying water is the water placed over sediment in the test vessel.

Pore water or interstitial water is the water occupying space between sediment and soilparticles.

Spiked sediment is sediment to which test chemical has been added.

Stabilization period is the interval between the addition of the spiked sediment to test vesselsand the addition of test organisms to the test vessels.

23ANNEX 2RECOMMENDATIONS FOR CULTURE OF FRESHWATER AMPHIPODS1. Hyalella azteca and other freshwater amphipod species including Gammarus pulex, G.

pseudolimnaeus, and G. fossarum must be cultured in a controlled temperature laboratoryfacility. All equipment, containers, and accessories that might contact the organisms or culturewater within the culturing facility must be clean, rinsed as appropriate, and made of non-toxicmaterials (e.g., glass, Teflon, nylon, and stainless steel). Culture water should be renewedroutinely and aerated gently to maintain the DO at 80% to 100% of saturation. The temperatureof culture water should be 23 ± 1°C for H. azteca and 18 ± 1°C for Gammarus sp., as a dailyaverage. Additionally, the instantaneous temperature of the culture water should be 23 ± 3°Cand 18 ± 3°C. A 16-h light and 8-h dark photoperiod is used.

2. Sources of culture water may be uncontaminated supply of groundwater, surface water, orreconstituted water for both H. azteca and Gammarus sp.. Acceptable water must allowsatisfactory survival, growth, and reproduction of the test species. For H. azteca, several recipesof reconstituted freshwater have been proposed. Among them, five-salt reconstituted water(SAM-5S) is recommended (Borgmann 1996). The following recipe provides reconstitutedwater within a hardness of 120 to 140 mg CaCO3/L, 0.8 mg Br/L, 1.8 mg Cl/L, conductivity of300 to 500 μS/cm, and pH of 6.5 to 8.5 and has been suitable for culturing H. azteca asdemonstrated in many laboratories (Environment and Climate Change Canada, 2017). TheCa:Br ratio shall be kept constant since these are essential for H. azteca and must be presenttogether (Borgmann, 1996). Na and HCO3 are the most essential ions for H. azteca survival,and Mg and K are needed for optimal growth. To prepare 40 L of SAM-5S reconstitutefreshwater, use reagent grade chemicals (anhydrous salts) as follows:✓ To 100 mL of highly purity distilled deionized or reverse osmosis water in a glass beakeradd the following:⚫ Calcium chloride (CaCl2): 4.44 g⚫ Sodium bicarbonate (NaHCO3): 3.36 g⚫ Magnesium Sulphate (MgSO4): 1.20 g⚫ Potassium chloride (KCl): 149 mg⚫ Sodium bromide (NaBr): 41.2 mg✓ Stir the contents of the beaker until all of the salts are dissolved.

✓ Place about 20 L of high purity deionized or distilled water in a clean container.

24✓ Pour the contents of the beaker (i.e., 100 mL water containing the salts) into the container,ensuring the entire contents of the beaker is transferred, and fill the container to 40 L withdeionized or distilled water.

✓ Aerate the mixture for at least 24 h at room temperature before use.

3. Substrates such as gauze strips, nylon mesh, plastic mesh, uncontaminated leaves, orshredded paper towels are placed in each culture chamber and replaced necessary.

4. A single ration diet such as commercial fish food flakes (e.g., TetraFin, TetraMin, andZeigler) or a mixed diet such as yeast- cerophyl and trout chow (YCT) are provided to thecultures. The amount of food distributed to each culture chamber will vary with the age andnumber of the amphipods in the chamber.

ReferencesASTM (2020) Standard Test Method for Measuring the Toxicity of Sediment-AssociatedContaminants with Freshwater Invertebrates: E1706-20. ASTM International, WestConshohocken, PA, USA.

USEPA (2016b) OCSPP 850.1020: Gammarid Amphipod Acute Toxicity Test. EPA712/C/16/012. October 2016.

Borgmann U (1996) Systematic analysis of aqueous ion requirements of Hyalella azteca: Astandard artificial medium including the essential bromide ion. Archives Environ ContamToxicol 30(3): 356-363.

Environment and Climate Change Canada (2017) Biological test method: test for survival andgrowth in sediment using the freshwater amphipod Hyalella azteca. Third Edition. RM.33.

September 2017. 25ANNEX 3PREPARATION OF FORMULATED SEDIMENTSediment compositionThe composition of the formulated sediment should be as follows:Constituent Characteristics Dry weight ratioQuartz sandGrain size: > 50% of the particles should be in therange of 50-200 μm75−76%Kaolin Kaolinite content 30% 20%PeatSphagnum moss peat, as close to pH 5.5-6.0 aspossible, no visible plant remains, finely ground(particle size d 1 mm) and air dried.

4−5%CaCO3 Pulverized, chemically pure 0.05−1%Water Water used for the toxicity test 30−50%PreparationThe peat is air dried and ground to a fine powder. A suspension of the required amount of peatpowder in deionized water is prepared using a high-performance homogenizing device. The pHof this suspension is adjusted to 5.5 ± 0.5 with CaCO3. The suspension is conditioned for atleast two days with gentle stirring at 20 ± 2 °C, to stabilize pH and establish a stable microbialcomponent. pH is measured again and should be 6.0 ± 0.5. Then the peat suspension is mixedwith the other constituents (sand and kaolin clay) and deionized water to obtain a homogeneoussediment with a water content in a range of 30−50% of dry weight of the sediment. The pH ofthe final mixture is measured once again and is adjusted to 6.5 to 7.5 with CaCO3 if necessary.

Samples of the sediment are taken to determine the dry weight and the organic carbon content.

Organic carbon content of the final mixture should be 2% (± 0.5%) and is to be adjusted by theuse of appropriate amounts of peat and sand.

StorageThe dry constituents for preparation of the artificial sediment may be stored in a dry and coolplace at room temperature. The formulated (wet) sediment should not be stored prior to its usein the test. It should be used immediately after the aging period.

ReferencesOECD Guideline 218 (2004) Sediment-Water Chironomid Toxicity Using Spiked Sediment26OECD Guideline 207 (1984) Earthworm, Acute Toxicity Test.

27ANNEX 4SEX DETERMINATION OF HYALELLA AZTECAAdult male Hyalella azteca can be identified by enlarged second gnathopods (Figure A4-1).

The absence of the enlarged gnathopods and the presence of developing eggs can be used topositively identify females (Figure A4-1). However, amphipods not identified as male areassumed to be female even if eggs are not present.

Figure A4-1. Photos of adult Hyalella azteca after 42 days of chronic toxicity test. (a) A maleamphipod with enlarged gnathopods and (b) a female with developing eggs. a)b)gnathopodeggs28ANNEX 5MASS BALANCE CALCULATIONS FOR SEDIMENT-WATER TOXICITYTESTS; EXAMPLE FOR SPIKED SEDIMENT TEST PERFORMED WITHFRESHWATER AMPHIPODS1. The starting point for a mass balance calculation for a study with sediment-dwellerorganisms in sediment-water tests using spiked sediment is the measured concentrations of thechemical in the test system considering the concentrations at the beginning and end of the studyas well any additional intermediate concentrations available.

2. The mass balance calculation in the case of studies with sediment-dwellers with spikedsediment is illustrated below for one test chemical stable in sediment and one test chemicalunstable in sediment such as two pesticides with different characteristics.

3. The information on the analytical determination of the chemical needed for the massbalance calculation is given for the one example in Table A5-1. Now we have only onemeasurement data that can be shown here, thus we will add another chemical data afterperforming experiments. The measurements in this case were at three time points (2 hours and28 days after starting exposure). For simplicity, they are reported here only at the lowest andhighest concentrations tested. However, measurements at additional tested concentrations arerecommended, particularly in the case of the chemicals that are difficult to test (e.g., whenconcentrations are poorly maintained in the test system).

4. For the two examples below, the test system contains an amount of sediment of 90 g-wet (55 mL) and a volume of overlying water of 220 mL; it is assumed that the pore water hasa volume of 9 mL. Based on this information, it is possible to calculate the amount of chemicalthat partitions to the various compartments by multiplying the concentration by thevolume/amount of water/sediment and to calculate the percentage in each compartment withrespect to that initially contained in the spiked sediment.

5. In order to assess the fate of the test chemical in the sediment/water system, the massbalance calculation was performed for each time point for which measurements were available(see Tables A5-2a and Tables A5-2b).

29Example 1: Test chemical stable in sedimentTable A5-1: Measured concentrations of the test chemical in different compartments of aspiked sediment study with Hyalella azteca under flow-through condition.

Measured concentrationsNominal(µg/kg)2 hours 28 daysOverlyingwater(ng/L)Pore water(ng/L)Sediment(µg/kg)Overlyingwater(ng/L)Pore water(ng/L)Sediment(µg/kg)Control

Table A5-2: Example of mass balance calculations for the test chemical.

Amounts and proportions calculated from measured concentrationsNominal(µg)2 hours 28 daysOverlyingwater(ng)Pore water(ng)Sediment(ng)Overlyingwater(ng)Pore water(ng)Sediment(ng)Control

6. In the example 1, the test chemical was measured almost exclusively in the sediment atthe start of exposure (i.e. more than 99.9 %), which was expected since the sediment had beenspiked prior to the start of the experiment. At the end of the exposure (i.e. after 28 days), theproportion of the test chemical in the sediment decreased to 62–70 % of that initially measured.

30The proportions of the test chemical in both overlying and pore water were below 0.2% over28 days likely due to extremely high hydrophobicity of this chemical. Although this experimentwas performed under a flow-through condition at the rate of 2 volumes addition per day, thelow concentration in overlying water led to negligible discharge outside the test beakerSpecifically, the discharge over 28 days accounted for less than 1% of the initially measuredamounts. Therefore, the reduction in sediment concentrations during exposure might beattributed to degradation rather than discharge outside the test beaker.

Example 2: Test chemical unstable in sedimentNot yet performed. We will add this example after obtaining the experimentalresult.