Dihexyl fumarate

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: 24 July 2018. Experimental completion date: 12 October 2018.

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:

yes

Remarks:

Only one set of samples from range finding were taken and stored frozen until required for analysis. Definitive test parental daphnids were fed with GM300 suspension replacing Tetramin. Deviations didnt affect the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control, solvent control and each test group from the bulk test preparation at 0 hours and 24 hour fresh and from the pooled replicates at 24 hour old and 48 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 48 hours and stored frozen for further analysis if necessary. The chemical analysis was conducted by Residue Analytical Services at Envigo – Eye.
All samples bottles were fortified by the addition of 5 mL of acetonitrile in order to stabilize the test item.

On receipt at Envigo - Eye, each sample was given a unique reference number and was stored at approximately -20°C, unless analysed immediately. When required for analysis, liquid samples were allowed to thaw pending analysis.

Vehicle:

yes

Details on test solutions:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010 and 0.10 mg/L.
A nominal amount of test item (20 mg) was dissolved in dimethylformamide (DMF) and the volume adjusted to 20 mL to give a 1.0 mg/mL solvent stock solution from which additional solvent stock solutions at 0.10 and 0.010 mg/mL were prepared. An aliquot (200 µl) of each of the solvent stock solutions was separately dispersed in 2 liters of test water with the aid of magnetic stirring for approximately 5 minutes to give the required test concentrations of 0.0010, 0.010 and 0.10 mg/L.
In the range finding test five daphnids were placed in each test and control vessel 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 and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test, control and solvent control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control and solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL of DMF.
A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. All samples were analyzed.

Definitive Test
Based on the results of the range finding test the following test concentrations were assigned to the definitive test 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.
A nominal amount of test item (20 mg) was dissolved in DMF and the volume adjusted to
20 mL to give a 1.0 mg/mL solvent stock solution from which a series of stock dilutions were made to give further solvent stock solutions of 0.56, 0.32, 0.18 0.10 mg/mL. An aliquot (200 µL) of each of the solvent stock solutions was separately dispersed in 2 liters of test water with the aid of magnetic stirring for 5 minutes to give the required test concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System and Supporting Information
The test was carried out using first instar Daphnia magna derived from in house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 2) 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 GM 300 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:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

20 - 21°C

pH:

7.8 - 8.0

Dissolved oxygen:

8.6 - 9.1 mg O2/L

Nominal and measured concentrations:

0.001, 0.01, 0.1 mg/L (range finding - nominal)
0.003, 0.0028, 0.0033, 0.0049, 0.0062 mg/L (definitive test - geometric mean)

Details on test conditions:

Exposure Conditions
As in the range finding test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test, control and solvent control vessel at random, in the test preparations. Four replicate test, control and solvent 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 and solvent control was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100µL/L of DMF.
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.

Assessments
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 Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.006 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 tests are given in Table 1.
No immobilization was observed at the test concentrations of 0.0010 and 0.010 mg/L, however, a single immobilized Daphnia was observed at 0.10 mg/L and two immobilized Daphnia were observed in the control.
Sub lethal effects of exposure were observed at 0.10 mg/L. This response was reduced mobility at both 24 and 48 hours and trapping at the surface at 24 hours.
Based on this information test concentrations of 0.010, 0.018, 0.032, 0.056 and 0.10 mg/L were selected for the definitive test.
Chemical analysis of the 0.010 and 0.10 mg/L test preparations at 0 hours showed measured test concentrations of 0.0038 and 0.054 mg/L (38% and 54% of nominal concentrations) respectively. Analysis of the 0.0010 mg/L test preparation at 0 hours showed a measured test concentration of less than the LOD.
Analysis of the old test preparations at 48 hours showed measured test concentrations had declined, to less than the LOD determined to be 0.0020 mg/L, in all test samples indicating instability of the test item under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Analysis of the fresh test preparations at 0 and 24 hours showed measured test concentrations to range from 0.0090 to 0.0934 mg/L (60% to 101% of nominal concentrations). Analysis of the 0.010 mg/L fresh preparation gave a measured concentration of 0.030 mg/L (301% of nominal) was obtained. This high value was considered to be due to a possible dosing error during preparation.
Analysis of the old test preparations at 24 and 48 hours showed measured test concentrations had declined, to less than the LOD determined to be 0.0010 mg/L. This decline was in line with the results obtained from the study to determine the Abiotic Degradation: Hydrolysis as a Function of pH (Envigo Study Number XP76GX) whereby approximately 50% degradation occurred after 324 hours at pH 4 and 7. Given this decline in measured test concentrations it was considered justifiable to base the results 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 to be 0.0030, 0.0028, 0.0033, 0.0049 and 0.0062 mg/L
The geometric mean measured test concentrations were determined to be:
Geometric Mean Measured Test Concentrastion (nominal) = 0.0030 (0.010), 0.0028 (0.018), 0.0033 (0.032), 0.0049 (0.056), 0.0062 (0.10) mg/L
As the nominal 0.010 mg/L test group was below the level at which any significant biological effects were observed, the possible error during dosing was considered to have had no impact on the outcome of the test.

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 2.
Inspection of the immobilization data at 24 and 48 hours based on the geometric mean measured test concentrations gave the following results:
24h: EC50 >0.0062 mg/L
48h: EC50 >0.0062 mg/L
The No Observed Effect Concentrations after 24 and 48 hours exposure were 0.0062 and 0.0049 mg/L, respectively. The Lowest Observed Effect Concentrations after 48 hours exposure was 0.0062 mg/L.

Sub-Lethal Effects
Sub lethal effects of exposure were observed at 0.0049 and 0.0062 mg/L. These responses were reduced mobility and covered with debris (see Table 2).

Validation Criteria
The test was considered to be valid given that none of the control and solvent 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 3.
Temperature was maintained at 20 °C to 21 °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 914 to 985 Lux.

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

Results with reference substance (positive control):

Positive Control
A positive control (Envigo study number KC41PP) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 25 September 2018 to 27 September 2018.
Exposure conditions for the positive control were similar to those in the definitive test.
In the positive control test at 0 and 48 hours the light intensity measured was in excess of the range specified in the study plan (500 to 1000 lux), the maximum light intensity recorded was 1037 lux. Given that the recorded light intensity was only marginally in excess of the specified range and as the daphnids in the control group did not show any mortalities or sub-lethal effects, this deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using Weibull analysis using linear maximum likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:
24H: EC50=1.1mg/L (95%CL:0.95-1.3mg/L), NOEC=0.56mg/L, LOEC=1.0 mg/L
48H: EC50=0.80mg/L (95%CL:0.12-1.5mg/L), NOEC=0.56mg/L, LOEC=1.0 mg/L

A 20% immobilization was recorded in the 0.32 mg/L test concentration; however, as the immobilization recorded in the 0.56 mg/L test concentration was only 10% immobilization it was considered unlikely that effect observed was as a result of exposure to the reference item and therefore the NOEC was 0.56 mg/L.
The NOEC 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.

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

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative ImmobilizedDaphnia		Observations		Cumulative ImmobilizedDaphnia		Observations
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	1	0	4N	5 N	1	1	4 N	4 N
Solvent Control	0	0	5 N	5 N	0	0	5 N	5 N
0.0010	0	0	5 N	5 N	0	0	5 N	5 N
0.010	0	0	5 N	5 N	0	0	5 N	5 N
0.10	0	1	3 N 1 R 1 T	2 N 2 R+T	0	1	2 N 3 R	4 R

Rep    =Replicate

N      = Normal

R     = Reduced mobility

T      = Trapped at surface

Table 2       Cumulative Immobilization Data and Observations in the Definitive Test

Geometric Mean Measured Test Concentration (mg/L)	24 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
Solvent Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0030	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0028	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0033	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0049	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0062	0	0	1	0	1	5	4 N 1 R	5 N	4 N	2 N 3 R
Geometric Mean Measured Test Concentration (mg/L)	48 Hours
	Cumulative ImmobilizedDaphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
Solvent Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0030	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0028	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0033	0	0	0	0	0	0	5 N	5 N	5 N	5 N
0.0049	0	0	0	0	0	0	5 N	3 N 2 R	5 N	4 N 1 R+C
0.0062	0	1	1	2	4	20	2 N 3 R	3 N 1 R	3 N 1 R	1 N 2 R

Rep    =Replicate

N          = Normal

R     = Reduced mobility

C     = Covered with debris

Table 3       Water Quality Measurements

Geometric Mean Measured Test Concentration (mg/L)		0 Hours (Fresh Media)			24 Hours (Old Media)
		pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	Rep1	7.9	8.8	20	7.9	8.7	21
Solvent Control	Rep1	7.8	8.6	20	7.9	8.7	21
0.0030	Rep1	7.8	8.7	20	7.9	8.6	21
0.0028	Rep1	7.8	8.7	20	7.9	8.7	21
0.0033	Rep1	7.6	8.7	20	7.6	8.5	21
0.0049	Rep1	7.9	8.7	20	7.9	8.7	21
0.0062	Rep1	7.9	8.7	20	7.9	8.6	21
Geometric Mean Measured Test Concentration (mg/L)		24 Hours (Fresh Media)			48 Hours (Old Media)
		pH	mg O2/L	Temperature °C	pH	mg O2/L	Temperature °C
Control	Rep1	8.0	9.1	20	7.9	8.5	21
Solvent Control	Rep1	8.0	9.1	20	7.9	8.6	21
0.0030	Rep1	8.0	9.1	20	8.0	8.6	21
0.0028	Rep1	8.0	9.1	20	8.0	8.5	21
0.0033	Rep1	8.0	9.1	20	7.9	8.4	21
0.0049	Rep1	8.0	9.1	20	7.9	8.4	21
0.0062	Rep1	8.0	9.1	20	7.9	8.5	21

Rep       = Replicate

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and based on the geometric mean measured test concentrations gave the following results:
48h: EC50 >0.0062 mg/L, NOEC: 0.0049 mg/L, LOEC 0.0062 mg/L

Description of key information

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and based on the geometric mean measured test concentrations gave the following results:

48h: EC50 >0.0062 mg/L, NOEC: 0.0049 mg/L, LOEC 0.0062 mg/L