Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 03 February 2017 Experimental completion date: 07 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 2,4-Pentanedione, peroxide (CAS 37187-22-7)
Batch: 1609427920
Purity: 29.7% active ingredient
Physical state/Appearance: Clear colorless liquid
Expiry Date: 30 September 2018
Storage Conditions: Approximately 4 °C in the dark
Analytical monitoring:
yes
Details on sampling:
Range-finding test:
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the initial experiment were analyzed.

Initial experiment:
Samples were taken for chemical analysis on Days 0, 5, 10, 14 and 20 (fresh media) and Days 1, 6, 11, 15 and 21 (old media), however, no analysis was performed.

Main test:
Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 20 and of the expired test preparations on Days 1, 6, 13 and 21. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Range-finding Test
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter give a 100 mg/L stock solution from which a series of dilutions was made to give the required test series of 0.10, 1.0, 10 and 10 mg/L.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Initial Experiment
A nominal amount of test item (40 mg) was dissolved in test water and the volume adjusted to 2 liters give a 20 mg/L stock solution from which a series of dilutions was made to give the required test series of 0.80, 1.0, 2.0, 4.0 and 8.0 mg/L.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
On Days 0 to 5, a nominal amount of test item (500 mg) was dissolved in test water and the volume adjusted to 5 liters to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further test solutions of 6.25, 12.5, 25 and 50 mg/L.
On Days 6 to 20, a nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give a 100 mg/L stock solution which was also the highest test concentration. A series of dilutions was made from this stock solution to give the further test solutions of 6.25, 12.5, 25 and 50 mg/L.
The stock solution and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 5, 12 and 20 (fresh media) and Days 1, 6, 13 and 21 (old media)
Test organisms (species):
Daphnia magna
Details on test organisms:
Test System and Supporting Information
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 ºC. 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 Tetramin® 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 Water
The reconstituted water (Elendt M7 medium) used for the range finding test, initial experiment and definitive test was the same as that used to maintain the stock animals.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
The water hardness was observed to be in the range 244 to 290 mg/L as CaCO3 in the control and the highest surviving test group throughout the test.
Test temperature:
Temperature was maintained at approximately 21 ºC to 22 ºC throughout the test
pH:
there were no treatment related differences for oxygen concentration
Dissolved oxygen:
there were no treatment related differences for pH
Nominal and measured concentrations:
Range-finding Test
In the range-finding test Daphnia magna were exposed to a series of nominal concentrations of 0.10, 1.0 and 10 mg/L.

Initial Experiment
In the initial experiment Daphnia magna were exposed to a series of nominal concentrations of 0.80, 1.0, 2.0, 4.0 and 8.0 mg/L.

Definitive Test
Based on the results of the initial experiment, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item.
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 mg/L for a period of 21 days.
Mean Measured Fresh Test Concentration (mg/L): 3.4, 7.0, 14, 24, 42
Mean Measured Fresh Test Concentration (mg ai/L): 1.0, 2.1, 4.0, 7.1, 12

Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0, 5, 12 and 20 (see Annex 3) showed measured test concentrations to range from 3.1 to 42 mg/L (equivalent to 0.93 to 12 mg active ingredient (ai/)L). An increase in the measured concentration of the aged test preparations on Days 1, 6, 13 and 21 was observed to between 3.4 and 57 mg/L (equivalent to 1.0 to 17 mg ai/L). Inspection of the data could show no reason for the increase observed. Duplicate samples were analyzed which showed a similar pattern of results. Given that the duplicate samples had been stored for a prolonged period of time, they were used for information purposes only and not used for calculation purposes. It was considered appropriate to calculate the results based on the average measured concentration of the freshly prepared media. At the request of the Sponsor, results were also calculated based on an active ingredient concentration of 29.7%.

Details on test conditions:
Throughout the test the light intensity was observed to be in the range 368 to 829 lux.

Range-finding Test
In the range finding test, for each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group five replicate test vessels were prepared. The water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 10 days.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed on Days 3, 5 and 7. The adult daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
A sample of each test concentration was taken for chemical analysis on Days 0 and 3 in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the initial experiment were analyzed.

Initial Experiment
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 ºC with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed daily. The adult daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
Samples were taken for chemical analysis on Days 0, 5, 10, 14 and 20 (fresh media) and
Days 1, 6, 11, 15 and 21 (old media), however, no analysis was performed.

Definitive Test
Exposure Conditions
For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group ten replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 ºC with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were renewed daily throughout the test. The adult daphnia were transferred to fresh media by wide bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted before being discarded.
Each daphnid received approximately 5 to 15 µL of an algal suspension (Desmodesmus subspicatus) and approximately 20 µL of Tetramin® flake food suspension daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

Assessments
Test Organism Observations
On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental daphnia as compared with the controls.
Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilization criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
At the end of the test, the length of each surviving parent animal was determined.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
3.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
19 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
15 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
4.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation and reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: immobilisation and reproduction
Details on results:
Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L, however, immobilization was observed at 10 mg/L. Sub lethal effects of exposure were observed at test concentrations of 1.0 mg/L and above. These responses were parental daphnids that were observed to be pale in coloration.
Based on this information test concentrations of 0.80, 1.0, 2.0, 4.0 and 8.0 mg/L were selected for the initial experiment.
Chemical analysis of the freshly prepared 1.0 and 10 mg/L test preparations on Day 0 showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.53 mg/L and 4.4 mg/L were obtained respectively, and of the old or expired media on Day 3 showed that measured concentrations of less than the LOQ and 3.7 mg/L were obtained respectively, indicating that the test item was not stable under test conditions.

Initial Experiment
Based on the results of a preliminary range finding test, test concentrations of 0.80, 1.0, 2.0, 4.0 and 8.0 mg/L were selected for the definitive test.
No significant effect was observed at any of the concentrations employed in terms of adult mortality or young production, therefore a definitive test was conducted, employing a wider concentration range in order to obtain ECx/NOEC values.

Definitive Test
Based on the results of the initial experiment, test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

Lethal Effects on the Parental Generation (P1)
Mortality (immobilization) occurred predominantly at the highest test concentrations of 24 and 42 mg/L (7.1 and 12 mg ai/L respectively) resulting in 100% mortality by Day 2 and 1 respectively. Mortality was also observed at the test concentrations of 3.4, 7.0 and 14 mg/L (1.0, 2.1 and 4.0 mg ai/L) on Days 2, 10 and 18. However, statistical analysis of the mortality data using the Step-down Cochran Armitage test procedure showed that the observed mortalities in the 3.4, 7.0 and 14 mg/L (1.0, 2.1 and 4.0 mg ai/L) test groups were not significantly different (P≥0.05) when compared to the control group. As a result these mortalities were classed as inadvertent for the purposes of the statistical analysis.

Sub-lethal Effects on the Parental Generation (P1)
All daphnids were observed to be the same size and color as the control daphnids with the exception of a single daphnid in the 7.0 mg/L (equivalent to 2.1 mg ai/L) test group on Days 16 and 17 which was observed to be pale prior to its death on Day 18, and 8 daphnids in the 14 mg/L (equivalent to 4.0 mg ai/L) test concentration which were observed to be pale on Day 7. This was a transient effect only lasting for one day and was therefore considered not to have had an impact on the outcome of the test.
After 21 days the length of each surviving adult was determined.
The results showed that there were no statistically significant differences (P≥ 0.05) between the control and the 3.4, 7.0 and 14 mg/L test groups (equivalent to 1.0, 2.1 and 4.0 mg ai/L respectively) in terms of length of the daphnids after 21 days exposure to the test item. The 24 and 42 mg/L test group (equivalent to 7.1 and 12 mg ai/L respectively) data was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.

Effects on Reproduction
After 21 days there were no statistically significant reductions between the control and the 3.4, 7.0 and 14 mg/L test groups (equivalent to 1.0, 2.1 and 4.0 mg ai/L) in terms of the number of live young produced per adult. The 24 and 42 mg/L test groups (equivalent to 7.1 and 12 mg ai/L) showed a significant difference as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.

Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.
Young were first produced in the control test group on Day 7 of the test.
Due to the toxic effect of the test item the parental generation (P1) of the 24 and 42 mg/L test groups (equivalent to 7.1 and 12 mg ai/L) were eliminated prior to the production of young.
There were no unhatched eggs or dead young in all control and treatment groups surviving to maturation.

Lowest Observed Effect Concentration
The LOEC was 24 mg/L (equivalent to 7.1 mg ai/L) as this test group produced no young throughout the test duration given that all adults were eliminated prior to young production.

No Observed Effect Concentration
The NOEC was 14 mg/L (equivalent 4.0 mg ai/L) as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P ≥ 0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

The following ECx(immobilization) values based on the fresh measured concentration were calculated by Probit analysis using max. likelihood regression at 21 days:

Endpoint

Concentration (mg/L)

Concentration (mg ai/L)

Immobilization

EC10

5.6

1.7

 

95% confidence limits

Not determined due to nature of the data

Not determined due to the nature of the data

 

EC50

13

3.9

 

95% confidence limits

Not determined due to nature of the data

Not determined due to the nature of the data

NOEC

14

4.0

LOEC

24

7.1


NOEC =           No Observed Effect Concentration

LOEC =            Lowest Observed Effect Concentration

The following ECx(reproduction) values based on nominal test concentrations were calculated by the Linear Interpolation method at 21 days:

Endpoint

Concentration (mg/L)

Concentration (mg ai/L)

Reproduction

EC10

15

4.3

 

95% confidence limits

14.98 – 15.00

4.30 – 4.31

 

EC50

19

5.6

 

95% confidence limits

18.99 – 19.00

5.5 – 5.6

NOEC

14

4.0

LOEC

24

7.1


NOEC No Observed Effect Concentration

LOEC Lowest Observed Effect Concentration

Validation Criteria

The following validation criteria were achieved during the test:

 

Required

Actual

Control mortality

20%

10%

Mean number of live young per surviving adult (control group)

60 after 21 days

144

Coefficient of variation for control group*

25%

6%

No ephippia produced

0

0

Dissolved oxygen

>3 mg O2/L

≥ 7.7mg O2/L

pH (control group)

6 to 9
Variation
1.5

7.3 to 8.2
0.9

 


*      Based on total number of living offspring per parent animal alive at the end of the test

 Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in significant mortalities at the concentrations of 24 and 42 mg/L (equivalent to 7.1 and 12 mg ai/L respectively) resulting in 100% mortality by Days 2 and 1 respectively.
The 21 Day EC50 (immobilization) value, based on the average measured concentration of the freshly prepared test preparations, for the parental daphnia generation (P1) was calculated to be 13 mg/L (equivalent to 3.9 mg ai/L).
The 21 Day EC50 (reproduction) based on the average measured concentration of the freshly prepared test preparations was 19 mg/L (equivalent to 5.6 mg ai/L).
The NOEC and the LOEC based on the average measured concentration of the freshly prepared test preparations were 14 and 24 mg/L, respectively (equivalent to 4.0 and 7.1 mg ai/L).
Executive summary:

 Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2012) No 211, "Daphnia magna Reproduction Test" referenced as Method C.20 of Commission Regulation (EC) No. 440/2008.

Methods

Based on the results of a preliminary range‑finding test and an initial experiment, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L for a period of 21 days.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin®flake food suspension.

Results

Analysis of the fresh test preparations on Days 0, 5, 12 and 20 showed measured test concentrations to range from 3.1 to 42 mg/L (equivalent to 0.93 to 12 mg active ingredient (ai/)L). An increase in the measured concentration of the aged test preparations on Days 1, 6, 13 and 21 was observed to between 3.4 and 57 mg/L (equivalent to 1.0 to 17 mg ai/L). Inspection of the data could show no reason for the increase observed and hence it was considered appropriate to calculate the results based on the average measured concentration of the freshly prepared media.

Exposure of Daphnia magna to the test item gave the following results based on the average measured concentration of the freshly prepared media:

Endpoint

Concentration (mg/L)

Concentration (mg ai/L)

Immobilization

EC10

5.6

1.7

 

95% confidence limits

Not determined due to the nature of the data

Not determined due to the nature of the data

 

EC50

13

3.9

 

95% confidence limits

Not determined due to the nature of the data

Not determined due to the nature of the data

Reproduction

EC10

15

4.3

 

95% confidence limits

14.98 – 15.00

4.30 – 4.31

 

EC50

19

5.6

 

95% confidence limits

18.99 – 19.00

5.5 – 5.6

No Observed Effect Concentration

14

4.0

Lowest Observed Effect Concentration

24

7.1

 

Description of key information

The 21 Day EC50 and EC10 (immobilization) value, based on the average measured concentration of the freshly prepared test preparations, for the parental daphnia generation (P1) were calculated to be 13 mg/L and 5.6 mg/L, respectively (equivalent to 3.9 mg a.i./L and 1.7 mg a.i./L).

The 21 Day EC50 and EC10 (reproduction) based on the average measured concentration of the freshly prepared test preparations were 19 mg/L and 15 mg/L, respectively (equivalent to 5.6 mg a.i./L and 4.3 mg a.i./L).

The NOEC and the LOEC based on the average measured concentration of the freshly prepared test preparations were 14 and 24 mg/L, respectively (equivalent to 4.0 and 7.1 mg a.i./L).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
1.7 mg/L

Additional information