[No public or meaningful name is available]

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:

20th June 2016 to 20th January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Deviations:
- The temperature of the fresh control solution at the start of the range-finding test was 23.3°C instead of minimum of 23.5°C. The difference was negligible.
- The pH of the highest concentration in the range-finding test changed by more than one unit during each refreshment interval. This was test item related change and could not be prevented.
- The determination of the TOC concentration in the test medium was performed with Shimadzu TOC-Lversion 1.04 instead of Shimadzu TOC-Control V version 2.10 (Shimadzu, Kyoto, Japan). This was an equivalent alternative.

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control.

- Sampling frequency : One day before the start of exposure to check the functioning of the system. At the start, after 7, 14, 21, 28 and 32 days of exposure. In addition, samples were taken on day 0 from the control treatment in order to check for possible contamination.
- Volume of samples : 1.8 mL from the total solution freshly prepared and from the old solutions in each vessel.
- Storage of samples : Samples not analysed on the day of sampling were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS (semi-static range-finding test)
The standard test procedures required generation of test solutions, which should contain completely dissolved test item concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system was prevented. A correction was made for the active content (a.c.) of the test item (correction factor of 1.39 was applied).
Preparation started with a concentration of 100 mg a.c./L (139 mg test item/L) in ISO medium. A short period of magnetic stirring (10-16 minutes) was sufficient to completely dissolve the test item in medium. The lower test concentrations were prepared by diluting the highest concentration in test medium. All solutions were clear and colourless.

PREPARATION OF STOCK SOLUTIONS (flow-trough final test)
The standard test procedures required generation of test solutions, which should contain completely dissolved test item concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. precipitate or a film of the test item on the water surface). A correction was made for the active content of the test item. Stock solutions of 5 g a.c./L (6.95 g test item/L) were prepared in Milli-RO water. A short period of magnetic stirring (10-28 minutes) was sufficient to completely dissolve the test item in medium.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name ans strain: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque.
- Source: In house culture.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Ratio male/female: 1:2
- Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
- Feeding brood stock: Pelleted fish food (Cyprico Crumble Excellent (300-500 um), SDS Diets, United Kingdom).
- Time of fertilisation: Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.

FEEDING
- Embryonic phase: No feeding.
- Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48-hours old. Food was supplied ad libitum.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Post exposure observation period:

None

Hardness:

Hardness was measured at the start and the end of the test in all test groups.
Total hardness was between 196 and 214 mg calcium carbonate per litre and thus above 140 mg/l as prescribed by the study plan.

Test temperature:

Temperature was measured at the start of the test, at weekly intervals and at the end of the test in one replicate per group. In addition, temperature was recorded continuously in one of the control replicates.
The average temperature measured at weekly intervals in the controls and the treatment groups varied between 24.6 and 25.4°C. The temperature continuously measured in one of the replicates of the control was maintained between 24.5 and 25.3°C. Hence, temperature was within the range described in the study plan: 25 ± 1.5°C.

pH:

pH was measured at the start of the test, at weekly intervals and at the end of the test in one replicate per group. In addition, temperature was recorded continuously in one of the control replicates.
The pH varied between 7.2 and 7.9 during the test period, which was within the range described in the study plan (6.0-8.5).

Dissolved oxygen:

Oxygen concentrations were measured at the start of the test, at weekly intervals and at the end of the test in one replicate per group. In addition, temperature was recorded continuously in one of the control replicates.
Oxygen concentrations varied between 6.7 and 8.9 mg/l during the test period, which is > 60% of oxygen saturation at 25°C as prescribed by the study plan.

Nominal and measured concentrations:

Nominal test concentrations: 0.46, 1.0, 2.2, 4.6 and 10.0 mg a.c./L (0.64, 1.4, 3.1, 6.4 and 13.9 mg test item/L).
Measured test concentrations: 0.35, 0.79, 1.7, 4.0 and 9.8 mg a.c./L (0.49, 1.1, 2.4, 5.6 and 13.6 mg test item/L).

Details on test conditions:

TEST SYSTEM
- Test type: Flow-through, with continuous renewal of test media
- Test duration: 32 days
- Test vessels: Stainless steel vessels (~1.7 l).
- Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Dosing system: Exact volumes of the test item stock solutions in Milli-RO water were dosed with syringes via a computer-controlled system consisting of five dispensers (Gilson). The dosed volumes of the stock entered a mixing flask separately from the dilution water supply (adjusted ISOmedium). The dilution water was supplied applying flow-meters at a constant rate of 5 litres per hour. In the mixing flask the dosed volume and the dilution water were mixed under continuous stirring. Peristaltic pumps, set at a rate of 1 litre per hour, were used to divide the contents of the mixing vessels continuously and equally over four replicate test vessels containing the eggs/fish larvae/juvenile fish. The flow meters were calibrated before the start
of the exposure and weekly thereafter. The whole system was checked twice daily.
- Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
- Experimental design: The experiment (nominal day 0) started with 80 fresh and healthy fertilised fathead minnow eggs per test group. The fertilised eggs were randomly distributed and divided equally over four stainless steel vessels. Each vessel contained 20 eggs in 1.7 l test medium. The media were continuously renewed (flow-through).
- Light period: 16 h photo-period daily, between 522 and 608 lux.
- Euthanasia: At the end of the test the surviving larvae were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM
Adjusted ISO medium with a hardness of 180 mg CaCO3 per liter and a pH of 7.7 +/- 0.3.
The Total Organic Carbon was measured at the start of the test in blank-medium. TOC measurements showed a value of 0.34 mg C/L, which was according to the study plan (< 2 mg C/L).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Stage of embryonic development: Daily, from the beginning of the exposure
- Hatching and survival: Daily from nominal day 4, numbers of hatched larvae and dead embryos, larvae and juvenile fish were recorded. Dead embryos, larvae and juvenile fish were removed directly after recording.
- Criteria for death of eggs: particularly in the early stages, a marked loss of translucency and change in coloration, caused by coagulation and/or precipitating of protein, characterised by a white opaque appearance;
- Criteria for death of embryos: Absence of body movement and /or absence of heartbeat;
- Criteria for death of larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque coloration of the central nervous system and/or lack of reaction to mechanical stimulus.
- Abnormal appearance: Daily, abnormalities were recorded, e.g. hyperventilating, uncoordinated swimming, atypical quiescence and atypical swimming behaviour.
- Body weight: At the end of the test, all surviving fish were weighed on a replicate basis (blotted dry weight).
- Body length: At the end of the test, individual lengths from the surviving fish were measured.

RANGE-FINDING STUDY
See the section "any other information on materials and methods".

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 13.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: hatching success

Remarks on result:

other: >= 9.8 mg a.c./L

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 13.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Remarks on result:

other: >= 9.8 mg a.c./L

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

>= 13.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks on result:

other: >= 9.8 mg a.c./L

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

0.49 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: 0.35 mg a.i./L

Details on results:

- Measured concentrations : The actual concentrations were at the level of 67-103% of target. Because the deviation from the target was on some occasions more than 20%, the arithmetic mean measured concentrations were calculated. The measured concentrations were maintained within 11% of mean measured (CV – coefficient of variation).
- Embryonic survival, development and hatching: The overall survival of embryos at the end of hatching was 91% in the control treatment, which complies with the requirements of the guideline (i.e. at least 70%). In the test concentrations, hatching success ranged from 91 to 95% and was not related to the applied dose. There was no significant delay in the onset of hatching observed in this study. Statistical analyses confirmed that hatching was not significantly affected up to and including 9.8 mg a.c./L. Hence, the NOEC was 9.8 mg a.c./L (13.6 mg test item/L).
- Larval survival and development: The mean post-hatch larval survival was 96% in the control. Hence, the validity criterion for post-hatch survival of at least 75% was met. The survival rates ranged from 92 to 99%, but were not dose-related. Statistical analyses confirmed that larval survival was not significantly affected up to and including 9.8 mg a.c./L. Hence, the NOEC was 9.8 mg a.c./L (13.6 mg test item/L). A number of effects were observed in this study, but the severity and incidence were not related to the applied dose.
- Effects on larval growth: A statistically significant effect on body weight was observed at the mean measured concentration of 0.79 mg a.c./L (LOEC), therefore, the NOEC for body weight was set at 0.35 mg a.c./L (0.49 mg test item/L). There were no statistically significant differences for both body length between the control and the different test item treatments. Hence, the NOEC for growth (mean body length of larvae) equalled 9.8 mg a.c./L (13.6 mg test item/L).

Reported statistics and error estimates:

The following statistical procedures were used to determine the NOEC for hatching success and larval survival, body length and weight.

NOEC for embryonic and larval survival:
- Hatchability and post-hatch survival: Chi²-2 x 2 table test with Bonferroni Correction (α=0.05, one-sided greater).

NOEC for body length and body weight:
- Data distribution: Shapiro-Wilk´s Test
- Homogeneity of variance: Levene´s Test (with Residuals)
Differences between treatments and the control: Williams Multiple Sequential t-test Procedure (α=0.05, one-sided smaller) after trend analysis by Contrasts (Monotonicity of Concentration/Response).

All analyses were performed with ToxRat Professional 3.2.1. (ToxRat Solutions® GmbH, Germany).

Range-finding test

The results of the range-finding test are presented in Table 1. No significant mortality or clinical effects were observed in the control and the three lowest concentrations during the exposure. In the highest concentration only 25% of the introduced embryos survived the exposure. All of the surviving larvae from the highest concentration were less pigmented and one showed loss of equilibrium. The actual concentrations measured in the freshly prepared solutions were at the level of nominal (95-104%). Measured concentrations were at the level of 86-101% of initial after 24 hours of exposure and 36-98% at the end of the 48-hour refreshment period.

Table 1: Survival of fish embryos and larvae during the range-finding test

Test item Nominal concentration (mg/L)	Test item Nominal concentration (mg a.c./L)	No. of eggs	Survival on nominal day								Total survival (100%)
		Day 0	1	2	3	4	5	6	7	8
Control	Control	10 10	10 10	10 10	10 10	10 10	10 10	10 9	10 9	10 9	100 90
0.14	0.1	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10	100 100
1.39	1.0	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10	10 10	100 100
13.9	10.0	10 10	10 10	10 10	10 10	10 10	9 9	9 9	9 9	9 9	90 90
139.1	100.0	10 10	10 10	10 10	10 10	10 10	10 10	10 10	2 3	2 3	20 30

1.     Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

Full ELS test

The results of the analysis of the samples taken during the test period are described in Table 2. The actual concentrations were at the level of 67-103% of target. Because the deviation from the target was on some occasions more than 20%, the arithmetic mean measured concentrations were calculated. The measured concentrations were maintained within 11% of mean measured (CV – coefficient of variation).

Table 2: Measured and average exposure concentrations during the ELS test

Test item^1 Nominal target conc. (mg a.c./L)	Measured concentration (mg a.c./L); nominal day						Mean	StDev	%CV
	0	7	14	21	28	32
0.46	0.419	0.368	0.340	0.351	0.307	0.321	0.35	0.040	11
1.0	0.878	0.818	0.851	0.752	0.708	0.719	0.79	0.071	9.1
2.2	1.94	1.89	1.72	1.80	1.53	1.58	1.7	0.17	9.5
4.6	4.40	4.29	4.33	3.95	3.38	3.86	4.0	0.39	9.6
10	10.1	9.67	10.1	10.3	9.18	9.44	9.8	0.43	4.4

1.     Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
Mean – arithmetic mean, StDev – standard deviation, %CV – variation coefficient

Table 3 and Table 4 lists data on the survival of embryos and hatching of larva per vessel during the first week of exposure, respectively. The overall survival of embryos at the end of hatching was 91% in the control treatment, which complies with the requirements of the guideline (i.e. at least 70%). In the test concentrations, hatching success ranged from 91 to 95% and was not related to the applied dose. There was no significant delay in the onset of hatching observed in this study. Statistical analyses confirmed that hatching was not significantly affected up to and including 9.8 mg a.c./L. Hence, the NOEC was 9.8 mg a.c./L (13.6 mg test item/L).

Table 3: Survival of fish embryos of the control and the test item treated groups

Treatment (mg a.c./L) (measured)	Vessel	Number of eggs at start	Nominal day										% Embryonic survival
			1		2		3		4		5
			D	A	D	A	D	A	D	A	D	A
Control	A	20	0	20	1	19	0	19	0	15	-	-	95
	B	20	2	18	0	18	0	18	0	13	-	-	90
	C	20	1	19	0	19	0	19	0	16	-	-	95
	D	20	2	18	0	18	0	18	1*	14	-	-	85
0.35	A	20	0	20	0	20	0	20	0	3	-	-	100
	B	20	0	20	0	20	0	20	1	14	-	-	95
	C	20	1	19	2	17	0	17	0	7	-	-	85
	D	20	3	17	0	17	0	17	0	17	-	-	85
0.79	A	20	1	19	0	19	0	19	0	18	-	-	95
	B	20	0	20	0	20	0	20	0	3	-	-	100
	C	20	1	19	0	19	0	19	1	13	-	-	90
	D	20	0	20	2	18	0	18	0	7	-	-	90
1.7	A	20	0	20	0	20	0	20	0	16	-	-	100
	B	20	1	19	0	19	0	19	0	3	-	-	95
	C	20	0	20	0	20	0	20	0	17	-	-	100
	D	20	2	18	2	16	0	16	0	14	-	-	80
4.0	A	20	0	20	0	20	0	20	0	20	-	-	100
	B	20	2	18	0	18	0	18	0	9	-	-	90
	C	20	0	20	0	20	0	20	0	19	-	-	100
	D	20	0	20	1	19	1	18	0	12	-	-	90
9.8	A	20	0	20	0	20	0	20	0	19	-	-	95
	B	20	2	18	0	18	0	18	0	18	-	-	90
	C	20	0	20	0	20	0	20	0	18	-	-	100
	D	20	1	19	0	19	1	18	1	12	-	-	85

D = dead, A= alive, * - embryo was missing, - no embryo was present (all hatched or did not survive)

 

Table 4: Hatching and survival of larvae of the control and the test item treated groups

Treatment (mg a.c./L) (measured)	Vessel	Number of eggs at start	Nominal day								Hatching success (%)
			3		4		5^1	6^1	7
			A	D	A	D	D	D	A	D
Control	A	20	-	-	3	1	0	0	18	0	95
	B	20	-	-	5	0	0	0	18	0	90
	C	20	-	-	3	0	0	0	19	0	95
	D	20	-	-	3	0	0	0	17	0	85
0.35	A	20	-	-	17	0	0	0	20	0	100
	B	20	-	-	5	0	0	0	19	0	95
	C	20	-	-	10	0	0	0	17	0	85
	D	20	-	-	0	0	0	0	17	0	85
0.79	A	20	-	-	1	0	1	0	18	0	95
	B	20	-	-	17	0	0	0	20	0	100
	C	20	-	-	5	0	0	0	18	0	90
	D	20	-	-	11	0	0	0	18	0	90
1.7	A	20	-	-	4	0	0	0	20	0	100
	B	20	-	-	16	0	0	0	19	0	95
	C	20	-	-	3	0	0	0	20	0	100
	D	20	-	-	2	0	1	0	15	0	80
4.0	A	20	-	-	0	0	0	0	20	0	100
	B	20	-	-	9	0	0	0	18	0	90
	C	20	-	-	1	0	0	0	20	0	100
	D	20	-	-	6	0	0	0	18	0	90
9.8	A	20	-	-	1	0	0	1	18	0	95
	B	20	-	-	0	0	0	0	18	0	90
	C	20	-	-	2	0	0	0	20	0	100
	D	20	-	-	4	1	0	0	16	0	85

-= no hatched larvae, A= Alive, D=Dead, 1.Only mortalities were counted

 

Table 5 (body weight) and Table 6 (length) present the group mean values with the percentage effect at the end of the test. A statistically significant effect on body weight was observed at the mean measured concentration of 0.79 mg a.c./L (LOEC), therefore, the NOEC for body weight was set at 0.35 mg a.c./L (0.49 mg test item/L).

Table 5: Mean body weight (mg) and percentage of reduction at the end of exposure

Test item^1 Mean measured conc. (mga.c./L)		Mean		Std. Dev.		n		%Reduction
Control		75		2.9		4		-
0.35		71		4.3		4		5.4
0.79		67		7.0		4		11*
1.7		63		6.2		4		16*
4.0		63		2.2		4		15*
9.8		67		6.7		4		11*

1. Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
*-effect statistically significant

Table 6: Mean body length (mm) and percentage of reduction at the end of exposure

Test item^1 Mean measured conc. (mga.c./L)	Mean	Std. Dev.	n	%Reduction
Control	21	0.39	4	-
0.35	21	0.38	4	1.0
0.79	20	0.76	4	2.3
1.7	20	0.54	4	3.8
4.0	20	0.21	4	3.8
9.8	20	0.61	4	1.9

1. Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

Acceptability of the test

1. The dissolved oxygen concentration was maintained above 60% of the air saturation value throughout the test;

2. The water temperature did not differ by more than ± 1.5°C between test chambers between successive days at any time during the test, and was within the temperature ranges specified for the test species;

3. Analytical results demonstrated that the concentrations of the test item in solution were generally maintained within ± 20% of the mean measured values. Effect parameters were based on average exposure concentrations.

4. Overall survival of fertilised eggs in the controls was >70% until hatching was complete (i.e. 91%). The overall survival of hatched larvae was >75% in the controls during the remaining test period (i.e. 96%).

Validity criteria fulfilled:

yes

Remarks:

Validity criteria of the OECD TG 210. See section "any other information on results"

Conclusions:

The present study assessed the possible lethal and sub-lethal effects of Reaction mass of N- [2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid during the embryonic and early larval development of the fathead minnow. The results led to the following conclusions:
1. Test item did not significantly affect hatching success of fathead minnow embryos at concentrations up to and including 9.8 mg a.c./L or 13.6 mg test item/L (NOEC).
2. Test item did not significantly affect post-hatch survival at concentrations up to and including 9.8 mg a.c./L or (13.6 mg test item/L (NOEC).
3. Test item did not significantly affect body length at concentrations up to and including 9.8 mg a.c./L or 13.6 mg test item/L (NOEC).
4. Test item significantly affected growth of fathead minnow (body mass) at a mean measured concentration of 0.79 mg a.c./L or 1.1 mg test item/L (LOEC).
The overall NOEC is 0.35 mg a.c./L (0.49 mg test item/L).

Executive summary:

A fish early-life stage toxicity test was performed with reaction mass of N-[2-(2-oxoimidazolidin-1 -yl)ethyl]methacrylamide and methacrylic acid (flow-through). The study procedures were based on the OECD guidelines for Testing of Chemicals: Guideline No. 210, 2013. In addition, the procedures were designed to meet the test methods of the OPPTS 850.1400, 'Public Draft', EPA 712-C-96-121, April 1996.

The batch of reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid tested was an amber liquid with an active content (a.c.) of 71.9% and completely soluble in test medium at the concentrations tested. The test was performed using a flow-through system with target concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg a.c./L (after correction for active content), which were based on the results of a preceding range-finding test. A control was also included.

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions were prepared in Milli-Q water. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The test was performed with four replicates containing 20 eggs of fathead minnow (Pimephales promelas) per replicate for each concentration and control. The test started by placing fertilised eggs in stainless steel test vessels. During the embryonic and larval phases, the eggs/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test (day 32), the surviving fish were measured and weighed. Samples for chemical analysis of the actual test item concentrations were taken one day before the start, at the start, at weekly intervals and at the end of the test. The actual concentrations were at the level of 67-103% of target. Because the deviation from the target was, on some occasions, more than 20%, the arithmetic mean measured concentrations were calculated and were 0.35, 0.79, 1.7, 4.0 and 9.8 mg a.c./L at the target concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg a.c./L (i.e. 76-98% of target).

The study met the acceptability criteria prescribed by the study plan and was considered valid. The results led to the following conclusions:

1. Test item did not significantly affect the hatching success of fathead minnow embryos at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC).

2. Test item did not significantly affect post-hatch survival at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC).

3. Test item did not significantly affect body length at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC)

4. Test item significantly affected growth of fathead minnow (body mass) at a mean measured concentration of 1.1 mg test item/L or 0.79 mg a.c./L (LOEC).

The overall NOEC is 0.49 mg test item/L (0.35 mg a.c./L).

Description of key information

 A study performed according to the OECD TG 210 and under GLP is available. Test item did not significantly affect the hatching success of fathead minnow embryos at concentrations up to and including 9.8 mg a.c./L or 13.6 mg test item/L (NOEC). Test item did not significantly affect post-hatch survival at concentrations up to and including 9.8 mg a.c./L or 13.6 mg test item/L (NOEC). Test item did not significantly affect body length at concentrations up to and including 9.8 mg a.c./L or 13.6 mg test item/L (NOEC). Test item significantly affected growth of fathead minnow (body mass) at a mean measured concentration of 0.79 mg a.c./L or 1.1 mg test item/L (LOEC). The overall NOEC is 0.35 mg a.c./L (0.49 mg test item/L).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.49 mg/L

Additional information

One reliable key study is available for this endpoint. The purpose of this study was to estimate the possible lethal and sub-lethal effects of reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid during the embryonic and early larval development of the fathead minnow. This study was conducted according to the OECD guideline 210 "Fish, Early-life Stage Toxicity Test" and according to GLP.

The batch of reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid tested was an amber liquid with an active content (a.c.) of 71.9% and completely soluble in test medium at the concentrations tested. The test was performed using a flow-through system with target concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg a.c./L (after correction for active content), which were based on the results of a preceding range-finding test. A control was also included.

In the flow-through system the dilution water was dosed separately from the test item stock solutions into mixing vessels. Stock solutions were prepared in Milli-Q water. The dosed volumes of stock and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily. The test was performed with four replicates containing 20 eggs of fathead minnow (Pimephales promelas) per replicate for each concentration and control. The test started by placing fertilised eggs in stainless steel test vessels. During the embryonic and larval phases, the eggs/larvae were observed for survival and effects on development, appearance and swimming behaviour. At the end of the test (day 32), the surviving fish were measured and weighed. Samples for chemical analysis of the actual test item concentrations were taken one day before the start, at the start, at weekly intervals and at the end of the test.

The actual concentrations were at the level of 67-103% of target. Because the deviation from the target was, on some occasions, more than 20%, the arithmetic mean measured concentrations were calculated and were 0.35, 0.79, 1.7, 4.0 and 9.8 mg a.c./L at the target concentrations of 0.46, 1.0, 2.2, 4.6 and 10 mg a.c./L (i.e. 76-98% of target).

The study met the acceptability criteria prescribed by the study plan and was considered valid. The results led to the following conclusions:

1. Test item did not significantly affect the hatching success of fathead minnow embryos at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC).

2. Test item did not significantly affect post-hatch survival at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC).

3. Test item did not significantly affect body length at concentrations up to and including 13.6 mg test item/L or 9.8 mg a.c./L (NOEC)

4. Test item significantly affected growth of fathead minnow (body mass) at a mean measured concentration of 1.1 mg test item/L or 0.79 mg a.c./L (LOEC).

The overall NOEC is 0.49 mg test item/L (0.35 mg a.c./L).