3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 04 April 2016 - Experimental completion date: 12 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical state/Appearance: White solid
Storage Conditions: Approximately 4 ºC in the dark

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the pooled replicates of the old or expired media at 24 and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A saturated solution was prepared by stirring an excess (100 mg/L) of test item in test water for a period of 48 hours prior to removing any undissolved test item present by filtration (0.2 µm filter, first approximate 2 liters discarded in order to pre-condition the filter) to give a saturated solution of the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

other: Reconstituted water (ISO medium)

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Temperature was maintained at 21 °C to 22 °C throughout the test

pH:

7.4 to 8.1 (see Table 5)

Dissolved oxygen:

8.6 mg O2/L to 9.5 mg O2/L (see Table A)

Nominal and measured concentrations:

Test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution

Details on test conditions:

Exposure Conditions:
As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated. The control group was maintained under identical conditions but not exposed to the test item. Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.

Test Organism Observations:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria:
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-Hour old test preparations. The pH and dissolved oxygen concentration were measured using a handheld meter whilst the temperature was measured using digital thermometer. The light intensity during the light period was measured using a Lux meter.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, the positive control was conducted between 07 September 2015 and 23 September 2015

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

4.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test:
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. No immobilization was observed at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution, however, immobilization was observed at 100% v/v saturated solution. Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test. Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.71 to 7.2 mg/L. A decline in measured concentrations was observed at 48 hours in the range of 0.39 to 4.5 mg/L indicating that the test item was not stable under test conditions. It was therefore considered appropriate to conduct the definitive test using a semi-static testing regime with media renewal after 24 hours exposure.

Verification of Test Concentrations:
Chemical analysis of the freshly prepared test samples at 0 and 24 hours showed measured test concentrations to range from 0.52 to 4.8 and from 0.53 to 5.4 mg/L respectively. A decline in measured test concentrations was observed in the old or expired media at 24 and 48 hours in the range of 0.37 to 3.5 mg/L. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined (see Table 2).

Immobilization Data:
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 4. Analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results: see Table 3. The No Observed Effect Concentration after 24 and 48 hours exposure was 2.1 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 4.2 mg/L. The slope and standard error of the response curves at 24 and 48 hours were 23.7 (SE = 1.4).

Sub-Lethal Effects:
Sub-lethal effects of exposure were observed in the 32 and 56% v/v saturated solution test concentrations. These responses were reduced mobility and trapping at the surface (see Table 4).

Validation Criteria:
The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria:
The results of the water quality measurements are given in Table 5. Temperature was maintained at 21 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH. Throughout the test the light intensity was observed to be in the range 562 to 614 Lux.

Observations on Test Item Solubility:
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test.Analysis of the immobilization data by the probit analysis using the linear maximum likelihood regression method at 24 and 48 hours using a specific computer software package based on the nominal test concentrations gave the following results: Table 6.
The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

Immobilization Data: The slope and standard error of the response curves at 24 and 48 hours were 23.7 (SE = 1.4).

Table 1:            Cumulative Immobilization Data and Observations in the in the Range-finding Test

Nominal Concentration (% v/v Saturated Solution)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	10 N	0	10 N
10	0	10 N	0	10 N
100	10	A/I	10	A/I

Table 2: Verification of Test Concentrations

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the Average Fresh Measured Test Concentration
10	0.44	85
18	0.77	85
32	1.2	79
56	2.1	76
100	4.2	82

Table 3: Immobilization Data

Time (h)	EC50(mg/L)
24	2.5
48	2.5

Table 4:            Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Concentration(mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.44	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.77	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.2	0	0	0	0	0	0	5 N	1 R 4 N	1 R 4 N	2 T 3 N
2.1	0	0	1	0	1	5	5 N	5 R	1 T 3 N	5 N
4.2	5	5	5	5	20	100	A/I	A/I	A/I	A/I

 

Geometric Mean Measured Concentration(mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.44	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.77	0	0	0	0	0	0	5 N	5 N	5 N	5 N
1.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
2.1	0	0	1	0	1	5	5 N	5 R	4 N	5 N
4.2	5	5	5	5	20	100	A/I	A/I	A/I	A/I

Table 5:            Water Quality Measurements 

Geometric Mean Measured Concentration(mg/L)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.4	9.1	21	7.4	9.0	21
0.44	R1	7.8	9.0	22	7.8	8.9	22
0.77	R1	7.9	8.9	22	7.9	8.9	21
1.2	R1	8.0	8.9	22	7.9	8.8	21
2.1	R1	8.1	8.8	22	8.0	8.8	21
4.2	R1	8.1	8.6	22	8.0	8.8	21

 

Geometric Mean Measured Concentration(mg/L)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	T°C	pH	mg O2/L	T°C
Control	R1	7.6	9.5	21	7.4	9.2	21
0.44	R1	7.7	9.4	22	7.8	9.1	21
0.77	R1	7.7	9.3	22	7.9	9.1	21
1.2	R1	7.8	9.2	22	8.0	9.1	21
2.1	R1	7.9	9.2	22	8.0	9.0	21
4.2	R1	7.9	9.0	22	8.1	9.0	21

R₁= Replicate 1

Table 6: Positive Control

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
24	0.90	Not possible to determine			0.56	1.0
48	0.54	0.47	-	0.63	0.32	0.56

 

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item has been investigated and gave the following results based on the geometric mean measured test concentrations:
Time point (hours) EC50 (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
48 2.5 2.1 4.2

Executive summary:

The EC50, No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) of ES421 Pinyl Nitrile are respectively 2.5 mg/L, 2.1 mg/L and 4.2 mg/L according to OECD Test Guideline 202 using the short-term toxicity to aquatic invertebrates method.

Description of key information

The EC50, No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) ofES421 Pinyl Nitrile are respectively 2.5 mg/L, 2.1 mg/L and 4.2 mg/L according to OECD Test Guideline 202 using the short-term toxicity to aquatic invertebrates method.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.5 mg/L