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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 February 2019 - 18 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 1812211800R
- Expiration date of the lot/batch: 21 December 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.10, 0.22, 0.45, 1.0, 2.2, 4.5 and 10 mg/L
- Sample storage conditions before analysis: The test samples were analyzed on the day of sampling.
- Water samples were taken from the control and each surviving test group (replicates pooled) for immediate quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 19 and of the expired test preparations on Days 2, 7, 14 and 21. Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (definitive test)
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 5 liters to give a 10 mg/L stock solution. This preparation was stirred using a magnetic stirrer for approximately 15 minutes to ensure adequate mixing and homogeneity. A series of dilutions was then made from this stock solution to give test solutions of 0.10, 0.22, 0.45, 1.0, 2.2 and 4.5 mg/L.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Stage and instar at study initiation: 1st instar
- Method of breeding: parthenogenesis
- Source: from in-house laboratory cultures
- Feeding during test : yes
- Food type: a mixture of algal suspension (Pseudokirchneriella subcapitata) and GM300 flake food suspension
- Amount: between 5 and 20 µL of an algal suspension; 20 µL of GM300
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
232 to 248 mg/L
Test temperature:
20 °C to 21 °C
pH:
7.9 ±0.3
Dissolved oxygen:
≥7.4 mg O2/L
Nominal and measured concentrations:
For the definitive test: nominal concentrations of 0.10, 0.22, 0.45, 1.0, 2.2, 4.5 and 10 mg/L
The geometric mean measured test concentrations were determined to be 0.085, 0.20, 0.37, 0.81, 1.7, 3.6 and 9.1 mg/L (77 to 91% of nominal concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL glass vessels
- Type: closed (sealed)
- Material, size, headspace, fill volume: completely filled with test preparation
- Aeration: no
- Renewal rate of test solution: The test preparations were renewed three times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19.
- No. of organisms per vessel: single daphnid per vessel
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Reconstituted Water – Elendt M4 Medium

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days.
- Light intensity: 668 to 1071 lux

EFFECT PARAMETERS MEASURED:
Immobilization
Reproduction
Body Length

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
No immobilization was observed at a test concentration of 0.10 mg/L, however, 20% immobilization was observed in the control and at 1.0 and 10 mg/L and 100% immobilization was observed at 100 mg/L. No sub lethal effects of exposure were observed at a test concentration of 0.10 mg/L. Sub lethal effects of exposure were observed at test concentrations of 1.0, 10 and 100 mg/L. These effects comprised a reduction in size (smaller), pale appearance and reduced mobility.
The number of neonates produced in the first brood in the control and at the test concentrations of 0.10 and 1.0 mg/L were considered to be comparable; however, no neonates were produced at test concentrations of 10 and 100 mg/L.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
9.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
3.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
5.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Body Length
Key result
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
10.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Body Length
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 9.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Body Length
Remarks on result:
other: Not possible to calculate 95% confidence limits for the data set.
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Body Length
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Body Length
Details on results:
- Verification of Test Concentrations:
Analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 0.077 to 9.8 mg/L (76% to 113% of the nominal test concentrations except on one occasion when a measured concentration of 137% of nominal was determined). A small decline in measured test concentration of the aged test preparations on Days 2, 7, 14 and 21 was observed to be between 0.068 and 8.3 mg/L (to 83% to 94% of the fresh measured test concentrations). Given these results (as not all values were within 20% of the nominal 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.

- Lethal Effects on the Parental Generation (P1):
Mortality (immobilization) occurred predominantly at the highest test concentration of 9.1 mg/L resulting in 100% mortality by Day 2.
Mortality was also observed at the test concentrations of 0.085, 0.37 and 3.6 mg/L. However, statistical analysis of the mortality data using Step-down Cochran-Armitage Test Procedure showed that the observed mortalities in the 0.085, 0.37 and 3.6 mg/L test groups were not significantly different (P > 0.05) when compared to the control group.
No mortalities occurred in the control and at the 0.20, 0.81 and 1.7 mg/L test groups throughout the test.

- Sub-lethal Effects on the Parental Generation (P1):
No sub lethal effects of exposure were observed in the control and at the test concentration of 0.085, 0.20, 0.37, 0.81 and 1.7 mg/L; however, at 3.6 mg/L one of the parental generation Daphnia was observed to be pale.
There was no significant effect on the size and color of the daphnids at 9.1 mg/L prior to observing 100% mortality by Day 2.
The results showed that there were no statistically significant differences (P >0.05) between the control and 0.085, 0.20, 0.37, 0.81 and 1.7 mg/L test group; however, a significant difference (P<0.05) was determined between the control and 3.6 mg/L test group in terms of reduction in length of the daphnids after 21 days exposure to the test item. The 9.1 mg/L test group data was not included in this analysis as exposure to the test item eliminated all of the daphnids prior to Day 21 of the test.

- Effects on Reproduction:
After 21 days there were no statistically significant differences (P> 0.05) between the control and the 0.085, 0.20, 0.37, 0.81 and 1.7 mg/L test groups. The 3.6 and 9.1mg/L test groups showed a statistically significant difference (P<0.05) from the control after 21 days in terms of producing fewer numbers of live young per adult.

- 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, all "filial" daphnids produced in the 0.81 mg/L test group were observed to be of normal appearance, whereas, in the control four out of 50 broods of neonates were observed to be pale in color, at 0.085 mg/L one out of 41 broods of neonates were observed to be pale in color, at 0.20 mg/L one out of 50 broods of neonates were observed to be pale in color, at 0.37 mg/L one out of 47 broods of neonates were observed to be pale in color, at 1.7 mg/L one out of 50 broods of neonates were observed to be pale in color and at 3.6 mg/L one out of 45 broods of neonates were observed to be pale in color.
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 9.1 mg/L test group died prior to the production of young.
There were no unhatched eggs or dead young observed in the control or any of the treatment groups throughout the test.






Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Results from the control and each test group were compared using the Multiple Sequential t-test incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution

The following validation criteria were achieved during the test:

 

Required

Actual

Control mortality

<20%

0%

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

 >60 after 21 days

158

Coefficient of variation for control group*

<25%

5.1%

No ephippia produced in the controls

0

0

Dissolved oxygen

>3 mg O2/L

≥7.4 mg O2/L

pH (control group)

6 to 9
Variation<
1.5

7.6 to 8.1
Variation 0.5


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

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item resulted in 100% mortality at a test concentration of 9.1 mg/L during the test period.
The 21 Day EC50 (immobilization) value, based on the geometric mean measured concentrations for the parental Daphnia generation (P1) was estimated to be 4.9 mg/L (with 95% confidence limits of 0.59 to 41 mg/L).
The 21 Day EC50 (reproduction) based on the geometric mean measured concentrations was 4.5 mg/L (with 95% confidence limits of 2.2 to 9.1 mg/L).
The 21-Day EC50 (body length) based on geometric mean measured concentrations was greater than >9.1 mg/L.
The LOEC and the NOEC based on the geometric mean measured concentrations were 3.6 and 1.7 mg/L respectively.
Executive summary:

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.

Based on the results of a preliminary range finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.10, 0.22, 0.45, 1.0, 2.2, 4.5 and 10 mg/L for a period of 21 days. The test solutions were renewed three times per week throughout the test. The test item solutions were prepared by dissolving a nominal amount of test item (50 mg) in test water and adjusting the volume to 5 liters to give a 10 mg/L stock solution. This test preparation was stirred using a magnetic stirrer for approximately 15 minutes to ensure adequate mixing and homogeneity. This solution was then further diluted as necessary, to provide the remaining test groups. The test was conducted in completely-filled and sealed vessels to minimize the potential for losses of the test item by volatilization.

The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension and supplemented with GM300 fish food suspension on Days 0, 1, 2 and 3.

Analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 0.077 to 9.8 mg/L (between 76 and 113% of nominal except on one occasion when a measured level of 137% of nominal was determined). A small decline in measured test concentration of the aged test preparations on Days 2, 7, 14 and 21 was observed to be between 0.068 and 8.3 mg/L (to between 83 and 94% of freshly prepared media concentrations). Given these results 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 to be 0.085, 0.20, 0.37, 0.81, 1.7, 3.6 and 9.1 mg/L (77 to 91% of nominal concentrations).

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Endpoint

Concentration mg/L
(95% confidence limits)

Immobilization

EC10

0.81 (0.02 to 34)

EC20

1.7 (0.12 to 24)

EC50

4.9 (0.59 to 41)

No Observed Effect Concentration

3.6

Lowest Observed Effect Concentration

9.1

Reproduction

EC10

3.3 (2.6 to 4.2)

EC20

3.7 (3.4 to 4.1)

EC50

4.5 (2.2 to 9.1)

No Observed Effect Concentration

1.7

Lowest Observed Effect Concentration

3.6

Body Length

EC10

5.6 (3.1 to 10)

EC20

10.6 (4.4 to 24)

EC50

>9.1 (not determined)*

No Observed Effect Concentration

1.7

Lowest Observed Effect Concentration

3.6


*Not possible to calculate 95% confidence limits for the data set.

Description of key information

Exposure of Daphnia magna to Butylal gave the following results based on the geometric mean measured test concentrations:

Endpoint

Concentration mg/L
(95% confidence limits)

Immobilization

EC10

0.81 (0.02 to 34)

EC20

1.7 (0.12 to 24)

EC50

4.9 (0.59 to 41)

No Observed Effect Concentration

3.6

Lowest Observed Effect Concentration

9.1

Reproduction

EC10

3.3 (2.6 to 4.2)

EC20

3.7 (3.4 to 4.1)

EC50

4.5 (2.2 to 9.1)

No Observed Effect Concentration

1.7

Lowest Observed Effect Concentration

3.6

Body Length

EC10

5.6 (3.1 to 10)

EC20

10.6 (4.4 to 24)

EC50

>9.1 (not determined)*

No Observed Effect Concentration

1.7

Lowest Observed Effect Concentration

3.6


*Not possible to calculate 95% confidence limits for the data set.

Key value for chemical safety assessment

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

Additional information

A determination of the chronic toxicity to aquatic invertebrates of butylal was conducted (Covance CRS Research Limited, 2020, LH48GK). The study was performed in accordance with EC method C.20 and OECD test guideline 211, and in compliance with GLP.

Following a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.10, 0.22, 0.45, 1.0, 2.2, 4.5 and 10 mg/L for a period of 21 days in Elendt M4 Culture medium at approx. 20 °C under static test conditions.

Analysis of the fresh and old test preparations showed decline in measured concentrations. Given these results (as not all values were within 20% of the nominal 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. 

Exposure of Daphnia magna to the test item resulted in 100% mortality at a test concentration of 9.1 mg/L during the test period.

The 21-Day EC50(immobilization) value, based on the geometric mean measured concentrations for the parental Daphnia generation (P1) was estimated to be 4.9 mg/L (with 95% confidence limits of 0.59 to 41 mg/L).

The 21-Day EC50(reproduction) based on the geometric mean measured concentrations was 4.5 mg/L (with 95% confidence limits of 2.2 to 9.1 mg/L).

The 21-Day EC50(body length) based on geometric mean measured concentrations was greater than >9.1 mg/L.

The LOEC and the NOEC based on the geometric mean measured concentrations were 3.6 and 1.7 mg/L respectively. 

Note that this NOEC is closed to the daphnid chronic value of 1.975 mg/L predicted by ECOSAR v1.11 (USEPA, 2012). This prediction is reliable since butylal is the log Kow and MW range of the model.