N-tert-butylacrylamide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: December 12, 2020 to Study Completion Date: April 17, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC

Details on sampling:

- Concentrations: 0 (control), 100 (100.04) mg/L
- Sampling method: Test item weighed for test concentration and formulation was prepared by mixing the test item in natural water in the aquarium to obtain the required concentration for the experiment.
- Sample storage conditions before analysis: sample was analysis immediately after preperation

Vehicle:

no

Details on test solutions:

500.2 mg of test chemical was weighed and dissolve in 5000 mL of natural water in a glass aquarium. Hence concentration of 100.04 mg/L were achieved, and control used was natural water

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Tarun Fish Farm, India
- Age at study initiation (mean and range, SD): Juveniles (originate from same source and population)
- Length at study initiation (length definition, mean, range and SD): 1.951 cm on average
- Weight at study initiation (mean and range, SD): 0.047 g on average

ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: yes Commercial fish food
- Feeding frequency during acclimation: Daily (except one day prior to the exposure).
- Health during acclimation (any mortality observed): Healthy

FEEDING DURING TEST
- Food type: no feed was given during study

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

170 mg CaCO3/L

Test temperature:

22.1-22.7 °C

pH:

6.5 to 7.1

Dissolved oxygen:

6.4-7.0 mg/L (77.46%-84.72%)

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal conccentration 0 and 100mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquarium
- Material, size, headspace, fill volume: glass aquarium 5 liters
- Aeration: no aeration during study
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1 replicate
- No. of vessels per control (replicates): 1 replicate
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.0246 g fish/L average

OTHER TEST CONDITIONS
- Photoperiod: 16 h light- 8 h dark
- Light intensity: 775 to 836 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Range finding study : Yes
- Test concentrations: 0 (control), 6.25, 12.5, 25, 50, 100 mg/L (with a geometric spacing factor of 2) in range-finding study
- Results used to determine the conditions for the definitive study: No mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0).

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no
- Observations on body length and weight: none
- Other biological observations:
- Mortality of control: no
- Other adverse effects control : no
- Abnormal responses:none

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

-

Sublethal observations / clinical signs:

Table1: Fish Mortality Data

Concentration (mg/L)	Mortality (number)									Cumulative Mortality(number) for period of 96 h
	Day 0		Day 1		Day 2		Day 3		Day 4
	2.5h	5h	24h	30h	48h	54h	72h	78h	96h
Range finding test
Control	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
12.5	0	0	0	0	0	0	0	0	0	0
25	0	0	0	0	0	0	0	0	0	0
50	0	0	0	0	0	0	0	0	1	1
100	0	0	0	0	0	0	0	0	0	0
Limit test
Control	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

Table2: LC₅₀Value

Parameters	96 hours
LC50	Greater than 100 mg/L
95% confidence interval- Lower Fiducial limit	NA
95% confidence interval - Upper Fiducial limit	NA

Key:LC₅₀- Median Lethal Concentration, NA- Not applicable

Appendix 1: Fish body weight and length record

S. No.	Body weight (g)	Length (cm)
Range finding test
1	0.026	1.951
2	0.037	1.965
3	0.035	1.975
4	0.054	1.890
5	0.037	1.832
6	0.036	1.929
7	0.031	1.803
8	0.038	1.894
9	0.025	1.815
10	0.035	1.948
Average	0.035	1.900
Limit test
1	0.048	1.960
2	0.050	1.873
3	0.034	1.889
4	0.052	1.992
5	0.058	1.985
6	0.045	1.978
7	0.068	1.997
8	0.038	1.975
9	0.035	1.964
10	0.037	1.892
Average	0.047	1.951

Table : Physicochemical Parameters (pH, Temperature (°C) and DO)

Range Finding Test
Hours	Parameters	Concentration (mg/L)
		Control	6.25	12.5	25	50	100
0	pH	7.0	7.0	6.8	6.8	7.0	7.0
	Temperature	23	23	23	22	23	23
	Dissolved oxygen	7.9	7.9	8.0	7.8	7.7	7.7
24	pH	7.1	6.9	6.9	6.8	6.8	7.0
	Temperature	23	23	23	23	23	22
	Dissolved oxygen	7.6	6.9	6.9	6.8	6.5	6.5
48	pH	7.1	7.0	7.0	6.5	6.6	6.4
	Temperature	23	23	22	23	23	23
	Dissolved oxygen	7.6	7.2	6.6	6.6	6.2	6.2
72	pH	6.9	6.9	6.5	6.5	6.5	6.4
	Temperature	23	23	23	23	23	23
	Dissolved oxygen	7.2	6.8	6.8	6.4	6.8	6.8
96	pH	6.9	6.5	6.4	6.4	6.2	6.5
	Temperature	23	22	22	23	23	23
	Dissolved oxygen	7.0	6.4	6.4	6.8	6.9	6.8
Limit test
Hours	Parameters	Concentration (mg/L)
		Control			100
0	pH	7.0			7.1
	Temperature	23			23
	Dissolved oxygen	7.0			6.4
24	pH	6.8			7.0
	Temperature	22			22
	Dissolved oxygen	6.8			6.5
48	pH	7.0			6.9
	Temperature	22			22
	Dissolved oxygen	6.6			6.9
72	pH	6.9			6.8
	Temperature	22			22
	Dissolved oxygen	6.6			6.8
96	pH	6.8			6.5
	Temperature	22			22
	Dissolved oxygen	6.9			6.6

Validity criteria fulfilled:

yes

Remarks:

No mortality in control were found throughout the 96-h test duration. Dissolve oxygen conc was maintained above 60% in all test vessels throughout the test. The recovery of active ingredient content was found between 80-120% up to 96h.

Conclusions:

Based on the effect on mortality of test fishes zebrafish (Danio rerio), the 96 h LC50 of test chemical was determined to be >100 mg/L.

Executive summary:

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.951 cm and 0.047g, respectively. Fish were fed (commercial fish food) daily (except one day prior to the exposure)during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 775-836 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 170 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22-23 °C,6.5 to 7.1, 6.4 to 7.0 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and the same was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 6.25, 12.5, 25, 50, 100 mg/L. During range finding test,no mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0). No mortality or abnormality was found in control group and 100 mg/L concentration. 7 fish were used/concentration during range finding and limit test. HPLC method was used for method validation and active ingredient analysis along with stability of the test chenical in the test medium. Test chemical was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during limit test 0 h and 96 h avg. recovery for 100 (100.04) mg/L conc were 96.21% & 96.10%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chenical to zebrafish (Danio rerio) was determined to be >100 mg/L. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

Description of key information

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.951 cm and 0.047g, respectively. Fish were fed (commercial fish food) daily (except one day prior to the exposure)during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 775-836 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 170 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22-23 °C,6.5 to 7.1, 6.4 to 7.0 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and the same was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 6.25, 12.5, 25, 50, 100 mg/L. During range finding test,no mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0). No mortality or abnormality was found in control group and 100 mg/L concentration. 7 fish were used/concentration during range finding and limit test. HPLC method was used for method validation and active ingredient analysis along with stability of the test chenical in the test medium. Test chemical was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during limit test 0 h and 96 h avg. recovery for 100 (100.04) mg/L conc were 96.21% & 96.10%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chenical to zebrafish (Danio rerio) was determined to be >100 mg/L. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

This study was conducted as per OECD 203 (2019) to assess the acute toxicity effects of test chemical on zebrafish (Danio rerio) following exposure up to 96 h under static condition. Juvenile fish of same age and normal in appearance were used in this (originate from same source and population). The average length and weight (10 fish) were observed 1.951 cm and 0.047g, respectively. Fish were fed (commercial fish food) daily (except one day prior to the exposure)during acclimatization. Photoperiod and light intensity were maintained such as16 h light- 8 h dark, 775-836 Lux during experiment. Hardness of water was measured once during acclimatization and found to be 170 mg CaCO3/L, temperature, pH and dissolve oxygen were maintained between 22-23 °C,6.5 to 7.1, 6.4 to 7.0 mg/L, respectively, throughout the test. Fish were acclimatized for 7 days prior dosing. Natural water was used as dilution control and the same was used for formulation of test chemical. The study was initiated with a range finding test by using following concentrations of 0 (control), 6.25, 12.5, 25, 50, 100 mg/L. During range finding test,no mortality or abnormality was found in control group as well as in all the tested groups. Hence, a limit test was conducted at 100 mg/L test concentration, along with a control group (0). No mortality or abnormality was found in control group and 100 mg/L concentration. 7 fish were used/concentration during range finding and limit test. HPLC method was used for method validation and active ingredient analysis along with stability of the test chenical in the test medium. Test chemical was found to be stable in the test medium. The active ingredient content results were considered acceptable (80-120%) as during limit test 0 h and 96 h avg. recovery for 100 (100.04) mg/L conc were 96.21% & 96.10%, respectively, were found in acceptable range. The test is valid as all the validity criteria are fulfilled: No mortality in control or vehicle control were found throughout the 96-h test duration; Dissolve oxygen conc. was maintained above 60% in all test vessels throughout the test; The recovery active ingredient content was found between 80-120% up to 96h. The 96 h LC50 of test chenical to zebrafish (Danio rerio) was determined to be >100 mg/L. Thus, test chemical was considered as non-toxic to aquatic fishes at environmental relevant concentrations and hence, considered to be not classified as per the CLP classification criteria.