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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:
01-06-2015 until 23-02-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
- see below
Principles of method if other than guideline:
During the definitive test the dissolved oxygen concentration, in the old media, for test concentration 4.09 mg/L was between 3.79 and 5.39 mg/L; however, on two occasions the dissolved oxygen was recorded as below the guideline criteria (>3 mg/L). This was thought not to have impacted the test as the parental daphnia all died by Day 7. The test area temperature range, for the duration of the test, exceeded the protocol criteria (+/- 2 °C) by 0.2 °C, however, the minimum/maximum temperature recorded per day did not exceed the protocol criteria. This was thought not to have impacted the test as the validity criteria were achieved.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: At Day 0, 6, 13 and 20 duplicate samples (ca. 10 mL) were taken from the preparation flasks for the control and each test concentration. For the old test media, at Day 1, 7, 14 and 21, the contents of the respective replicates were pooled prior to sampling. One sample was analysed and the second sample stored as a ‘back up’ should further analysis be required.
- Sample storage conditions before analysis: Due to the volatile nature of the test material, sample preparation for analysis was performed as quickly as possible.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Prior to the start of the test, and at each media renewal during the test, approximately 20 mg of test material was dissolved in a final volume of 2000 mL of Elendt M4 Media to give the initial 10 mg/L test solution. The test concentrations were then prepared by serial dilution of this initial solution.Once the daphnia in the top concentration had all died (Day 13) the test concentrations were prepared by adding ca. 6.4 mg of test material to 2000 mL of Elendt M4 media to give the 3.2 mg/L test solution. The remaining test concentrations were then prepared by serial dilution.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea- Strain/clone: Daphnia magna (Straus)
- Source: Parental laboratory cultures, held and maintained at the test facility. The culture was originally obtained from another site owned by the testing facility.
- Age of parental stock: At the start of the test, juvenile Daphnia magna were <24 hours old.
- Feeding during test: The Daphnia magna were fed daily with a suspension of Chlorella vulgaris.

ACCLIMATION
- Food type: The Daphnia magna were fed with a suspension of Chlorella vulgaris
- Amount: The daily feeding rate of Chlorella vulgaris was up to a maximum of 5 x 10^6 algal cells per mL, per day. The amount of algae added to each test vessel was based on the age of the Daphnia magna and on the amount of precipitated algae present on the base of the vessels, or in suspension, directly before feeding.
- Frequency: Daily

CULTURING OF DAPHNIA MAGNA
- The Daphnia magna are cultured in 1 litre glass beakers containing 800 mL of Elendt M4 medium. Each vessel and its contents are referred to as a 'culture'. New cultures are initiated with juvenile Daphnia magna (less than 24 hours old), at a density of approximately 15 daphnids per litre. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris.The water in each culture was renewed or partially renewed at least twice a week. Juveniles were removed when present in cultures using a sieve. Cultures were maintained up to a maximum of 4 to 5 weeks. Juveniles for use in acute toxicity tests were collected from the second brood onwards. Approximately 24 hours before a test was set up, juveniles present in the cultures were removed and discarded. Over the next 24 hours, juveniles for use in the test were removed from the culture using a wide bore pipette and transferred to fresh culture medium. The juveniles were then left for at least 1 hour before selecting actively swimming individuals for use. All juveniles used to initiate a test were less than 24 hours old.All cultures, prior to and during toxicity tests, are maintained under fluorescent lighting on a 16-hour light:8-hour dark photoperiod with an approximate 30 minute dawn/dusk period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
202 to 260 mg/L (as CaCO2)
Test temperature:
20 ± 2 °C
pH:
6.69 to 8.11
Dissolved oxygen:
2.28 to 9.78 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0, 0.1, 0.32, 1.0, 3.2 and 10 m/L
- Mean measured concentrations: 0, 0.0445, 0.0962, 0.224, 0.953 and 4.09 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: ca. 50 mL glass jars.
- Type: Sealed with lids to reduce losses through evaporation .
- Material, size, headspace, fill volume: ca. 50 mL.
- Aeration: No. The test water was aerated prior to the test until oxygen saturation was reached. During the test period, the test water was not aerated.
- Renewal rate of test solution: Media renewal at 24 hour intervals.
- No. of organisms per vessel: 1.
- No. of vessels per concentration (replicates): 10.
- No. of vessels per control (replicates): 10.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used in the study was Elendt M4 medium prepared in reverse osmosis water.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: The pH, dissolved oxygen concentration (% air saturation value (ASV) and mg/L) and temperature were determined in freshly prepared test media at the start of each renewal period and in pooled samples of the replicate test vessels at each test concentration at the end of each exposure period. Continuous temperatures were measured in an additional vessel maintained in the study arena.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 hours of light, 8 hours of darkness.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- At approximately 24-hour intervals, the adult Daphnia magna were observed for immobility, the presence or absence of eggs developing in the brood pouch (gravid or non-gravid) and mortality. In addition, the number of juveniles present (alive or dead) were recorded, removed from the vessels and then discarded.A Daphnia magna was considered immobile and therefore dead if, when the contents of the test vessel were briefly agitated, they did not swim during a 15-second period of observation.The appearance, colour and behaviour of the test substance in the test media were recorded at the start and end of the test and in each test media renewal interval.At the end of the test, the carapace lengths of all surviving parental Daphnia magna were measured using a microscope under low magnification fitted with a graticule slide.The carapace measurement was between the top of the head to the base of the apical spine. The apical spine was not measured as this can vary in length between moults.

VEHICLE CONTROL PERFORMED:
- No, no vehicle used.

RANGE-FINDING STUDY:
- No.A range-finding test was not conducted as information on the acute toxicity to Daphnia magna was available. The 48-hour NOEC for the acute Daphnia magna test was 5.6 mg/L.
Reference substance (positive control):
yes
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
1.775 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % CL 0.953 to 2.095 mg/L
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.953 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.953 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.953 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.953 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.953 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
The test preparations were observed to be colourless solutions throughout the duration of the test.

MORTALITY OF DAPHNIA MAGNA
- A summary of the parental Daphnia magna mortality during the definitive test is presented in a Table 1. If a Daphnia magna was considered immobile it was therefore considered dead and was removed from the test. Any replicate in which the parent Daphnia magna did not survive through to the end of the test was excluded from the analysis.Total parental Daphnia magna survival on Day 21 is presented in Table 2.No significant effects on the adult survival were observed at test concentration below 4.09 mg/L (time weighted mean measured concentration) when compared to the control treatment. Complete mortality was observed at 4.09 mg/L (time weighted mean measured concentration).The NOEC value for adult survival was considered to be 0.953 mg/L based on time weighted mean measured concentrations. The EC50 for adult survival was considered to be 1.775 mg/L based on time weighted mean measured concentrations.DAPHNIA MAGNA JUVENILE PRODUCTIONThe mean number of live juveniles produced per treatment by Day 21 from surviving parental Daphnia magna is presented in Table 3.Live juveniles were observed in the control and majority of concentrations by Day 7 and 8. By Day 9 all surviving adults in all concentrations had produced a first brood.Given these results, it was considered that the test material had no effect on the time to the first brood. Vessels were excluded from the statistical analysis if the parental Daphnia magna did not survive to Day 21. Any juveniles produced by these Daphnia magna were excluded from the analysis.In the control group, the mean number of young produced per Daphnia magna by Day 21 was 64; therefore the validity criterion for mean number of juveniles produced by the control group exceeding 60 at the end of the test was satisfied.The coefficient of variation (≤25%) for the mean number of juveniles produced per parent in the controls was 14.15%. No reproduction effects were observed on concentration 4.09 mg/L as the parent Daphnia magna died before producing juveniles.No significant negative effects on juvenile production were observed when compared to the control treatments. The juvenile numbers (per surviving adult) increased significantly as the concentration increased, up to 0.953 mg/L, indicating a potential positive test material effect.The NOEC value for juvenile production was considered to be 0.953 mg/L based on time weighted mean measured concentrations.The EC50 for juvenile production was considered to be greater than 0.953 mg/L based on time weighted mean measured concentrations.No aborted eggs were observed in the control or treatment groups throughout the test.The surviving parents did not produce any dead juveniles throughout the test with the exception of one control replicate which died on the same day as the 7 dead juveniles were observed and one replicate in concentration 0.0445 and 0.953 mg/L which each produced one dead juvenile during the test.

SURVIVING ADULT GROWTH MEASUREMENTS
- The mean carapace lengths at each treatment are presented in Table 4.No significant effects on the growth were observed at any surviving test concentration when compared to the control treatments.The NOEC value for growth was considered to be 0.953 mg/L based on time weighted mean measured concentrations.The EC50 for growth was considered to be greater than 0.953 mg/L based on time weighted mean measured concentrations.

TOXICITY VALUES
- The NOEC and LOEC and Day 21 EC10, 20 and 50 toxicity values in terms of growth, survival and juvenile production per surviving parental Daphnia magna were derived statistically and are presented in Table 5. Results were based on time weighted mean measured concentrations (mg/L).

VALIDITY CRITERIA
- The validity criteria were met. The observed values for parental mortality and mean number of juveniles produced per adult in the control were 10% and 64, respectively.

WATER QUALITY AND ENVIRONMENTAL CONDITIONS
- The dissolved oxygen concentration, in the old media, for test concentration 4.09 mg/L (time weighted mean measured concentration) was between 3.79 and 5.39 mg/L, however, on two occasions (Day 1 and Day 7) the dissolved oxygen was recorded as below the criteria of 3 mg/L. This was thought not to have impacted the test as the majority of the parental daphnia died on days 2, 3, 4 and 6 where the dissolved oxygen remained above the criteria (4.27, 3.79, 5.10 and 5.39 mg/L, respectively). There were no mortalities on Day 1 and there was one remaining daphnia which died on Day 7. Therefore, the decrease in dissolved oxygen was not thought to have contributed to the parental mortality observed.

CHEMICAL ANALYSIS
- Chemical analysis of the test preparations of fresh (new) media showed measured concentrations to range from 0 to 100% of nominal.Chemical analysis of the test preparations of old media showed measured concentrations to range from 0 to 55% of nominal.As measured concentrations were not maintained within the 80 to 120% range described in the OECD Guideline, the toxicity of the test material to Daphnia magna was expressed in terms of time weighted mean measured concentrations.As there was limited or no measured concentration recoveries in the corresponding old media, ½ the LOQ value (0.025 mg/L) was substituted into the time weighted mean measured concentration calculations when no test material recovery was available.The time weighted mean measured concentrations were 0.0445, 0.0962, 0.224, 0.953 and 4.09 mg/L, equivalent to 45, 30, 22, 30 and 41% of nominal concentrations, respectively.
Results with reference substance (positive control):
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity.
Reported statistics and error estimates:
Formal statistical analysis was conducted on the data using the statistical software package CETIS v1.8.6. The test concentration results were compared to the control results. All results were based on time weighted mean measured concentrations.The adult survival NOEC was calculated using the Fishers Exact/ Bonferroni-Holm Test.The adult growth and juvenile production NOECs were calculated using the Bonferroni Adjusted t Test.The adult survival, growth and juvenile production EC10, 20 and 50 values were calculated using Linear Interpolation (ICPIN).

Table 1: Mortality Levels

Day Number

Time Weighted Mean Measured Concentration (mg/L)

Control

0.045

0.0962

0.224

0.953

4.09

1

-

-

-

-

-

-

2

-

-

-

-

-

2

3

-

-

-

-

-

5

4

-

-

1

-

-

1

5

-

-

-

1

-

-

6

-

-

-

-

-

1

7

-

-

-

-

-

1

8

-

-

-

-

-

-

9

1

-

-

-

-

-

10

-

-

-

-

1

-

11

-

-

-

1

-

-

12

-

-

-

-

-

-

13

-

-

-

-

-

-

14

-

-

1

-

-

-

15

-

-

-

1

-

-

16

-

-

-

-

-

-

17

-

-

-

-

-

-

18

-

-

-

-

-

-

19

-

-

-

-

1

-

20

-

-

-

-

-

-

21

-

-

-

-

-

-

% Mortality after 21 days

10

0

20

30

20

100

 

Table 2: Total Parental Survival on Day 21

Time Weighted Mean Measured Concentration (mg/L)

Survival of parental Daphnia magna at Day 21 (%)

Control

90

0.0445

100

0.0962

80

0.224

70

0.953

80

4.09

0

 

Table 3: Juvenile Production

Time Weighted Mean Measured Concentration (mg/L)

Mean number of juvenile produced per parental Daphnia magna

Control

64

0.0445

76

0.0962

79

0.224

102

0.953

137

4.09

NA

 

Table 4: Growth Measurements

Time Weighted Mean Measured Concentration (mg/L)

Mean length of surviving parental Daphnia magna after 21 days (mm)

Control

4.0

0.0445

4.1

0.0962

4.1

0.224

4.1

0.953

4.1

4.09

NA

 

Table 5: Toxicity Values

Parameter

ECx (mg/L)

95% LCL (mg/L)

95% UCL (mg/L)

NOEC (mg/L)

LOEC (mg/L)

 

Adult survival

EC10

0.0770

0.0615

1.079

0.953

4.09

EC20

0.197

0.0788

1.297

EC50

1.775

0.953

2.095

Juvenile production

EC10

>0.953

NC

NC

0.953

NC

EC20

>0.953

NC

NC

EC50

>0.953

NC

NC

Growth

EC10

>0.953

NC

NC

0.953

NC

EC20

>0.953

NC

NC

EC50

>0.953

NC

NC

LCL = Lower 95% Confidence Limits

ULC = Upper 95% Confidence Limits

NC = Not Calculable

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the NOEC value for adult survival was considered to be 0.953 mg/L. The EC50 for adult survival was considered to be 1.775 mg/L. The NOEC value for juvenile production was considered to be 0.953 mg/L and the EC50 for juvenile production was considered to be greater than 0.953 mg/L. The NOEC value for growth was considered to be 0.953 mg/L and the EC50 for growth was considered to be greater than 0.953 mg/L. All values were based on time weighted mean measured concentrations.
Executive summary:

A 21-day study was conducted to determine the effects of exposure to the test material on Daphnia magna reproduction and survival in accordance with the standardised guideline OECD 211 under GLP conditions. The test was conducted using a semi-static design with renewal of the test media at 24 hour intervals. Ten replicate test vessels were prepared for each treatment and the control group. A single juvenile Daphnia magna (<24 hours old) was added to each test vessel.

Nominal concentrations of 0.1, 0.32, 1.0, 3.2 and 10 mg/L were used. The 10 mg/L concentration was prepared by direct addition of the test material to Elendt M4 medium and the remainder prepared by serial dilution. A control group, containing Elendt M4 medium only, was also prepared. Analysis of test media samples was conducted on Days 0, 6, 13 and 20 (fresh media) and Days 1, 7, 14 and 21 (corresponding old media). As measured concentrations were not within the 80 to 120% range, the toxicity of the test material to Daphnia magna was expressed in terms of time weighted mean measured concentrations. The time weighted mean measured concentrations were 0.0445, 0.0962, 0.224, 0.953 and 4.09 mg/L, equivalent to 45, 30, 22, 30 and 41% of nominal concentrations, respectively.

No significant effects on the adult survival were observed at test concentrations below 4.09 mg/L when compared to the control treatment. Complete mortality was observed at 4.09 mg/L. The NOEC value for adult survival was considered to be 0.953 mg/L. The EC50 for adult survival was considered to be 1.775 mg/L. No significant negative effects on juvenile production were observed when compared to the control treatments. The juvenile numbers (per surviving adult) increased significantly as the concentration increased, up to 0.953 mg/L, indicating a potential positive test material effect. The NOEC value for juvenile production was considered to be 0.953 mg/L. The EC50 for juvenile production was considered to be greater than 0.953 mg/L. No significant effects on the growth were observed at any surviving test concentration when compared to the control treatments. The NOEC value for growth was considered to be 0.953 mg/L. The EC50 for growth was considered to be greater than 0.953 mg/L.

The validity criteria for control parental mortality (≤20%) and juvenile production (≥60) were both met and therefore the test is considered valid.

Under the conditions of this study, the NOEC value for adult survival was considered to be 0.953 mg/L. The EC50 for adult survival was considered to be 1.775 mg/L. The NOEC value for juvenile production was considered to be 0.953 mg/L and the EC50 for juvenile production was considered to be greater than 0.953 mg/L. The NOEC value for growth was considered to be 0.953 mg/L and the EC50 for growth was considered to be greater than 0.953 mg/L. All values were based on time weighted mean measured concentrations.

Description of key information

NOEC and EC50 (adult survival) = 0.953 and 1.775 mg/L

NOEC and EC50 (juvenile production) = 0.953 mg/L and >0.953 mg/L

NOEC and EC50 (growth) = 0.953 and >0.953 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.953 mg/L

Additional information

A 21-day test was conducted in accordance with the standardised guideline OECD 211 under GLP conditions (Anderson, 2016) to study the reproduction and survival of the aquatic invertebrate Daphnia magna. The test was conducted using a semi-static design with renewal of the test media at 24 hour intervals. Ten replicate test vessels were prepared for each treatment and the control group. A single juvenile Daphnia magna (<24 hours old) was added to each test vessel. Analysis of test media samples was conducted on Days 0, 6, 13 and 20 (fresh media) and Days 1, 7, 14 and 21 (corresponding old media). As measured concentrations were not within the 80 to 120% range described in the guideline, the toxicity of the test material to Daphnia magna was expressed in terms of time weighted mean measured concentrations. The time weighted mean measured concentrations were 0.0445, 0.0962, 0.224, 0.953 and 4.09 mg/L, equivalent to 45, 30, 22, 30 and 41% of nominal concentrations, respectively.

The validity criteria for control parental mortality (≤20%) and juvenile production (≥60) were both met and therefore the test is considered valid. Under the conditions of this study, the NOEC value for adult survival was considered to be 0.953 mg/L. The EC50 for adult survival was considered to be 1.775 mg/L. The NOEC value for juvenile production was considered to be 0.953 mg/L and the EC50 for juvenile production was considered to be greater than 0.953 mg/L. The NOEC value for growth was considered to be 0.953 mg/L and the EC50 for growth was considered to be greater than 0.953 mg/L. All values were based on time weighted mean measured concentrations.