4,4'-methylenebis[2,6-diethylaniline]

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-01-2020 to 07-07-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test item concentrations in the freshly prepared and aged test media were analyzed from six renewal periods (one renewal period per week). From the first two renewal periods, the samples taken from the three highest test concentrations were analyzed. From the following four renewal periods, the highest concentration was omitted and the next two concentrations were analyzed as in the undiluted filtrate no larvae had hatched.

Vehicle:

no

Details on test solutions:

For preparation of the test media, the test item was weighed into test water at a loading rate of 100 mg/L. This loading rate was clearly above the solubility limit of the test item in test water of approximately 1.6 mg/L (determined in a pre-experiment under GLP). Following suspension of the test item, the suspension was sonicated to achieve maximum solubilisation. The undiluted test solution was prepared by filtering and combining the filtrates. The undiluted filtrate was used as highest test concentration and was diluted with adequate volumes of test water for preparation of the test media with the lower test concentrations, i.e. the dilutions 1:3.2, 1:10, 1:32, and 1:100. Additionally, a control was run in parallel.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The study was performed with newly fertilised eggs of zebrafish, Danio rerio (Hamilton-Buchanan 1822, Teleostei, Cyprinidae). The origin of the strain of zebrafish is IME (Fraunhofer Institute, 57392 Schmallenberg, Germany). At test lab, a brood batch of this strain is held in accordance with the Swiss Animal Protection Law under the lab Animal Breeding License Number CH-SO-002. The parental fish were held in an aquarium in reconstituted water. A 16-hour light to 8-hour dark photoperiod with a 30-min transition period was applied. The fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49324 Melle/Germany) and brine shrimps (Artemia salina). The brood batch was regularly visually checked for abnormal behaviour, diseases or mortality. No visible abnormalities were observed in the fish during the whole culturing period and no medication was applied.
Glass dishes covered with a stainless steel mesh were placed on the bottom of the aquarium one day prior to test start (Day –1) for collecting the newly fertilised eggs for the test. The mesh separated fertilised eggs (which sank to the bottom into the glass dishes) from the parent fish. Spawning started in the morning on Day 0 (start of the test = introduction of the eggs into the test media) after turning on the light. The eggs were collected from the glass dishes and were randomly exposed to the test media. Exposure to the test media started within 3 hours after fertilization

Test type:

semi-static

Water media type:

other: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations: CaCl2 × 2H2O: 147 mg/l - MgSO4 × 7H2O: 61.5 mg/l - NaHCO3: 32.5 mg/l - KCl: 2.9 mg/l

Remarks:

The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

Limit test:

no

Total exposure duration:

34 d

Post exposure observation period:

none

Hardness:

125 mg/l CaCO3 (1.25 mmol/l)

Test temperature:

25.8–26.9°C

pH:

7.3–7.6

Dissolved oxygen:

Dissolved oxygen concentration was 8.5 mg/l at the start (day 0) and 7.5-7.6 at the end of the study (day 34). During the test period, the lowest dissolved oxygen concentration measured in a single replicate of the control and at the test concentrations was 6.4 mg/L, corresponding to an oxygen saturation of at least 78%. Thus, dissolved oxygen concentration was sufficiently high throughout the test period, fulfilling the test guideline requirement of a 60% minimum.

Salinity:

Reconstituted water used

Nominal and measured concentrations:

Mean measured concentrations: 0, 0.077, 0.33 and 105 mg/L corresponding to the control, 1:10, 1:3.2 and the undiluted test substance suspension.

Details on test conditions:

The test vessels were placed into a temperature regulated water bath to keep the water temperature constant during the test period. The mean water temperature during the test period measured in the vessels of the different test concentrations and the control was in the range between 26.2 and 26.4°C. A 16 hour light to 8-hour dark photoperiod was used. Light intensity during the light period was in the range between 5.4–6.8 µmol s-1 m-2. The test media were not aerated until Day 25. Starting from Day 22 aeration was started to keep the oxygen concentration in the test media sufficiently high. The test duration was in total 34 days consisting of a hatching period from Day 0 to Day 4 plus a 30-day post hatch period. The Day 4 post fertilization equals Day 0 post hatch period. A semi-static test design with short test medium renewal intervals of 48 hours was considered for this study. The test item concentrations in the test media were kept as constant as possible during the test period and the water quality parameter were maintained at levels which meet the environmental requirements of the test fish. At the test medium renewal dates, the eggs, larvae and surviving fish were transferred into clean test vessels with freshly prepared test medium. Four replicates were used for each treatment (the test concentrations and the control). At the start of the test (Day 0), 80 fertilised eggs were randomly distributed to the replicate test vessels of each treatment (20 eggs per replicate). The fish loading rate of all replicates of all treatments at the end of the test period was calculated to be at a maximum of 94 mg fish wet weight/L.

Reference substance (positive control):

no

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

larval development

Key result

Duration:

34 d

Dose descriptor:

EC10

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

34 d

Dose descriptor:

LOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

100% mortality of eggs

Details on results:

Analysis of test item concentrations - The analytical measurements at the end of the renewal periods demonstrated a decrease of the test item concentrations during the test medium renewal periods. The decrease of test item concentrations during the renewal periods showed a high variation between the different treatments and is attributed to specific properties of the test item. The undiluted filtrate and the test media in the test vessels appeared to be clear solutions throughout the test period. The mean measured concentrations of the test item over the test period was determined to be 0.077, 0.33 and 1.5 mg/L corresponding to the 1:10, 1:3.2 and the undiluted suspension. The concentrations corresponding to the dilutions 1:32 and 1:100 were not measured since they were below the overall NOEC determined in this study.

2. Effect levels - The overall NOEC of the test item for early life stages of zebra fish was determined to be 0.33 mg/L (mean measured concentration) since no adverse effects were observed on the test fish up to and including this test concentration. The overall LOEC was determined to be 1.5 mg/L (mean measured concentration) due to the 100% mortality of eggs at this test concentration. The EC10 value for hatching rate was calculated to be 0.22 mg/L with 95% confidence limits from 0.21 to 0.23 mg/L. The LC10/EC10 values for survival rate, fish body length and fish weight (wet weight and dry weight) were determined to be >0.33 mg/L (mean measured) which was the highest test concentration where larvae had hatched.

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

No statistical evaluation was performed as the results did not indicate a toxic effect (no concentration effect relationship was observed and the hatching rate at the test concentrations was equal to the control).

Analytical results for test samples

Sampling Day / Sample Age	Dilution Factor of Undiluted Filtrate	Sample No.	Measured Concentration of test item	Sample Preparation Factor	Determined Concentration of Test Item	% from Initially Measured
			x		c
[d/h]	(Loading Rate 100 mg Test Item /L)		[mg/L]	F	[mg/L]	[%]
0/0	Control	F1	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F7	0.1002	1.25	0.125	-
	1:3.2-Diluted Filtrate	F9	0.356	1.25	0.446	-
	Undiluted Filtrate	F11	1.32	1.25	1.66	-
Feb 48	Control	F13	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F28	0.0248	1.25	0.031	25
	1:3.2-Diluted Filtrate	F33	0.243	1.25	0.304	68
	Undiluted Filtrate	F38	1.2	1.25	1.5	90
2/0	Control	F43	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F49	0.1005	1.25	0.126	-
	1:3.2-Diluted Filtrate	F51	0.303	1.25	0.379	-
	Undiluted Filtrate	F53	1.28	1.25	1.6	-
Apr 48	Control	F55	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F70	0.0499	1.25	0.0624	50
	1:3.2-Diluted Filtrate	F75	0.261	1.25	0.326	86
	Undiluted Filtrate	F80	1.1	1.25	1.38	86
10/0	Control	F121	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F127	0.11	1.25	0.138	-
	1:3.2-Diluted Filtrate	F129	0.355	1.25	0.444	-
Dez 48	Control	F133	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F145	0.0208	1.25	0.0259	19
	1:3.2-Diluted Filtrate	F149	0.133	1.25	0.166	37
20/0	Control	F189	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F195	0.108	1.25	0.134	-
	1:3.2-Diluted Filtrate	F197	0.388	1.25	0.485	-
22/48	Control	F201	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F213	0.0935	1.25	0.117	87
	1:3.2-Diluted Filtrate	F217	0.374	1.25	0.467	96
26/0	Control	F245	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F251	0.11	1.25	0.137	-
	1:3.2-Diluted Filtrate	F253	0.334	1.25	0.417	-
28/48	Control	F257	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F269	0.0375	1.25	0.0469	34
	1:3.2-Diluted Filtrate	F273	0.186	1.25	0.233	56
32/0	Control	F301	n.d.	1.25	< LOQ	-
(fresh)	1:10-Diluted Filtrate	F307	0.126	1.25	0.157	-
	1:3.2-Diluted Filtrate	F309	0.36	1.25	0.45	-
34/48	Control	F313	n.d.	1.25	< LOQ	-
(aged)	1:10-Diluted Filtrate	F325	0.0119	1.25	0.0149	9
	1:3.2-Diluted Filtrate	F329	0.0848	1.25	0.106	24

n.d. = no test item detected

n.a. = not applicable

LOQ = 0.00599 mg/L

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Further data as overall survival of fertilised eggs, body lengths etc. can be found in the attached PDF-document.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the overall NOEC of the test substance for early life stages of zebra fish was determined to be 0.33 mg/L. The EC10 for hatching rate was calculated to be 0.22 mg/L and the LC10/EC10 for survival rate, fish body length and fish weight (wet weight and dry weight) were determined to be >0.33 mg/L (Peither, 2020).

Executive summary:

A study was conducted to determine the toxicity of the test substance to the early life stages of zebra fish (Danio rerio) according to OECD Guideline 210 and OPPTS Guideline 850.1400, in compliance with GLP. Freshly fertilized eggs of zebra fish were exposed to the test media containing the test substance at dilutions of 0, 1:100, 1:32, 1:10, 1:1.32 and the undiluted test substance suspension in water under semi-static conditions for 34 days. At the start of the test, 80 eggs per treatment (divided into 4 replicates) were introduced in the test. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival. Only the three highest test concentrations (the undiluted filtrate and the dilutions 1:3.2 and 1:10) were analyzed, as the lower test concentrations were below the NOEC determined in this test. The mean measured concentrations of the test substance corresponding to the undiluted filtrate, 1:10 and 1:1.32 dilutions were detemined to be 1.5, 0.33 and 0.077 mg/L respectively. Under the study conditions, the overall NOEC of the test substance for early life stages of zebra fish was determined to be 0.33 mg/L. The EC10 for hatching rate was calculated to be 0.22 mg/L and the LC10/EC10 for survival rate, fish body length and fish weight (wet weight and dry weight) were determined to be >0.33 mg/L (Peither, 2020).

Description of key information

A study was conducted to determine the toxicity of the test substance to the early life stages of zebra fish (Danio rerio) according to OECD Guideline 210 and OPPTS Guideline 850.1400, in compliance with GLP. Freshly fertilized eggs of zebra fish were exposed to the test media containing the test substance at dilutions of 0, 1:100, 1:32, 1:10, 1:1.32 and the undiluted test substance suspension in water under semi-static conditions for 34 days. At the start of the test, 80 eggs per treatment (divided into 4 replicates) were introduced in the test. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival. Only the three highest test concentrations (the undiluted filtrate and the dilutions 1:3.2 and 1:10) were analyzed, as the lower test concentrations were below the NOEC determined in this test. The mean measured concentrations of the test substance corresponding to the undiluted filtrate, 1:10 and 1:1.32 dilutions were detemined to be 1.5, 0.33 and 0.077 mg/L respectively. Under the study conditions, the overall NOEC of the test substance for early life stages of zebra fish was determined to be 0.33 mg/L. The EC10 for hatching rate was calculated to be 0.22 mg/L and the LC10/EC10 for survival rate, fish body length and fish weight (wet weight and dry weight) were determined to be >0.33 mg/L (Peither, 2020).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.33 mg/L