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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:
2017
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
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Details on sampling:
Duplicate 10 mL measured samples of test media from the controls and each surviving test concentration were taken for analysis on days 0, 7, 14 and 20 from freshly prepared test media and on days 1, 8, 15 and 21 from old test media.
Vehicle:
no
Details on test solutions:
Water Source and Treatment
Water (Harrogate tap water supply) used for the holding of Daphnia stocks and for the toxicity tests with Daphnia magna enters the facility and passes through three activated carbon filters (in parallel) prior to sterilisation by a UV filter. The water is then heated or chilled to the required species specific temperature and supplied to the laboratories. Water was adjusted for hardness where required. Water quality is monitored twice a year, with a full microbial screen and trace pesticide residue analysis (performed by an external company).

Stock solution preparation
CDBC is not soluble and saturated solution preparation methods were necessary. An initial media preparation trial confirmed that a saturated solution, stirred for 72 hours, filtered (0.45µm) and then used to prepare the lower concentrations by serial dilution, was the most appropriate method for preparing the test solutions. However, a second media trial conducted after early termination of a first aborted in vivo test provided contradictory information (i.e., inconsistent concentration recovery or no recovery at all).
Further media work, conducted in conjunction with the 48-hour range-finding test supported the use of the saturated solution preparation method, stirring period of 72 hours, filtration (0.45µm Whatman Cellulose Nitrate Filter) and daily renewal of media, however, it also indicated that due to the low solubility and analytical method constraints it was not possible to determine reproducible analytical measurements for the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile Daphnia magna (less than 24 hours old) were taken from healthy laboratory cultures, held and maintained at Smithers Viscient (ESG) Ltd. The cultures were originally obtained from Smithers Viscient, Shawbury, UK. The Daphnia magna are cultured in 1000 mL glass beakers containing ~900 mL of treated mains water. 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 10-20 daphnids per litre. The cultures are fed daily with a concentrated suspension of Chlorella vulgaris prepared in accordance with standard operating procedures.
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 6 weeks (generally kept for 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.
Regular tests are conducted using a reference toxicant to ensure that Daphnia magna cultures are of the highest quality and sensitivity.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
Not reported.
Test temperature:
18.5 - 21.0 °C (min-max). Min/Max temperatures varied by more than 2°C, however, the temperature of the media at each renewal did not vary by more than 2°C at any time during the definitive test.
pH:
7.18 - 9.08 (min-max). The pH exceeded the range of 6-9 on one occasion, however this was not considered to impact the test as the validity criteria for parent mortality (=20%) and juvenile production (=60) were both achieved in the control.
Dissolved oxygen:
6.45 - 10.97 mg/L (min-max).
Salinity:
Freshwater.
Conductivity:
Not measured.
Nominal and measured concentrations:
Nominal: saturated solution (1 mg/L, i.e. around 1000 times the water solubility limit) serially diluted by a factor 3.2
Measured concentrations: See table 1.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Fill volume: 50 mL of solution, with a headspace
- Aeration: None.
- Renewal rate of test solution: every day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary
- Photoperiod: 16L:8D
- Light intensity:

EFFECT PARAMETERS MEASURED
- Parent immobilisation (monitored every day)
- Living neonate production per parent (monitored every day)
- Parental size (determined at the end of the experiment)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
no
Key result
Duration:
21 d
Basis for effect:
immobilisation
Remarks:
Parents
Remarks on result:
other: No adverse effect up to the water solubility limit.
Key result
Duration:
21 d
Basis for effect:
reproduction
Remarks on result:
other: No adverse effect up to the water solubility limit.
Key result
Duration:
21 d
Basis for effect:
growth
Remarks on result:
other: No adverse effect up to the water solubility limit.
Details on results:
Mortality of Daphnia magna
The parental Daphnia magna mortality during the definitive test is presented in Table 2. If a Daphnia magna was considered immobile it was therefore considered dead and was removed from the test. There was no dose-response relationship between parent mortality and treatment. No significant effects on the adult survival were observed at any test concentration when compared to the control treatments. The NOEC value for adult survival was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. In other words, it can be concluded that no significant effect was observed on parent Daphnia magna mortality up to the water solubility limit of CDBC.

Daphnia magna Reproduction
The numbers of juveniles produced in each test vessel over the duration of the test are presented in Table 3. Live juveniles were observed in the control and majority of test concentrations by Day 8. By Day 10 all surviving adults in the control test had produced a first brood. The mean number of young produced per control daphnia by Day 21 was 82; 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 was 22%. There was no dose-response relationship between reproduction and treatment. No significant effects on reproduction were observed at any test concentration when compared to the control treatments. The NOEC value for reproduction was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. In other words, it can be concluded that no significant effect was observed on parent Daphnia magna mortality up to the water solubility limit of CDBC.

Growth Measurements
The results of the individual carapace measurements are presented in Table 4. There was no dose-response relationship between growth and treatment. No significant effects on the growth were observed at any test concentration as compared to the control treatments. The NOEC value for growth was considered to be the 100 percent saturated solution of 1.0 mg/L nominal loading rate. In other words, it can be concluded that no significant effect was observed on parent Daphnia magna growth up to the water solubility limit of CDBC.
Results with reference substance (positive control):
None.
Reported statistics and error estimates:
Formal statistical analysis was conducted on the data using the statistical software package CETIS v1.8.6.8. The test concentration results were compared to the control results. All results were based on percent saturated solutions of nominal loading rate. The adult survival NOEC was calculated using the Fishers Exact/ Bonferroni-Holm Test. The adult growth and reproduction NOECs were calculated using the Bonferroni Adj Test. Adult survival, growth and reproduction data were statistically checked for dose-response relationship.

Table 1: Parent Daphnia magna survival at the end of the test.


Percent saturated solution of 1.0 mg/L
nominal loading rate

Survival of parental Daphnia magna at Day 21
(%)

Control

90

1.0

90

3.2

90

10

100

32

80

100

80

Table 2: Mean number of juvenile produced per parent Daphnia magna alive at the end of the test.


Percent saturated solution of 1.0 mg/L nominal loading rate

Mean number of juvenile produced per
parent Daphnia magna alive at the end of the test

Control

82

1.0

83

3.2

76

10

87

32

72

100

74

Table 3: Mean length of parent Daphnia magna alive at the end of the test.


Percent saturated solution of 1.0 mg/L
nominal loading rate

Mean length of surviving parental
Daphnia magnaafter 21 days
(mm)

Control

4.2

1.0

4.1

3.2

4.2

10

4.2

32

4.1

100

4.2

Validity criteria fulfilled:
yes
Conclusions:
No dose response and no significant effect were observed parent mortality or growth and reproduction. Therefore, it is concluded that CDBC has no long-term adverse effect on Daphnia magna up to its limit of water solubility.
Executive summary:

A 21-day Daphnia magna reproduction study was conducted under daily static renewal conditions to determine the effects of exposure to Bis(dibutyldithiocarbamato-S,S’)copper (CDBC) on Daphnia magna reproduction and survival in accordance with the OECD Chemicals Testing Guideline No.211 Daphnia magna Reproduction Test (October 2012). Based on the results of a 48 hour range-finding test (nominal concentrations 1.0 and 100  mg/L), which is not fully reported, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32, and 100% of a 1.0 mg/L loading rate. A control group was also included.

In an effort to maintain the test concentrations and maximize exposure to CDBC, the test was conducted as a daily static renewal design (including week-ends). Ten replicate test vessels were prepared for each treatment and control group. A single juvenile Daphnia magna (<24 hours old) was added to each test vessel.

Each day during the renewal process, parental animals were transferred into freshly prepared test media. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on each day was recorded. At the end of the test, carapace lengths of all surviving parental Daphnia magna were measured microscopically.

Due to the low solubility and analytical method constraints it was determined that it was not possible to determine reproducible analytical measurements for the test. The method used demonstrated that incurred samples had been exposed to the test substance but cannot be reliably quantified with the instrumentation currently available. Given the low solubility, solutions were prepared as saturated solutions and the analytical results, detailed in the below table, will only be reported as verification of dosing.

Mortality of Daphnia magna

No significant effects on the adult survival were observed at any test concentration when compared to the control treatments.

The NOEC value for adult survival was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on parent Daphnia magna mortality up to the water solubility limit of CDBC.

 

Daphnia magna Reproduction

No significant effects on the growth were observed at any test concentration as compared to the control treatments.

The NOEC value for growth was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on Daphnia magna reproduction up to the water solubility limit of CDBC.

 

Growth of Daphnia magna

No significant effects on the growth were observed at any test concentration as compared to the control treatments.

The NOEC value for growth was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on parent Daphnia magna growth up to the water solubility limit of CDBC.

No dose response and no significant effect were observed parent mortality or growth and reproduction. Therefore, it is concluded that CDBC has no long-term adverse effect on Daphnia magna up to its limit of water solubility.

Description of key information

A 21-day Daphnia magna reproduction study was conducted under daily static renewal conditions to determine the effects of exposure to Bis(dibutyldithiocarbamato-S,S’)copper (CDBC) on Daphnia magna reproduction and survival in accordance with the OECD Chemicals Testing Guideline No.211 Daphnia magna Reproduction Test (October 2012). Based on the results of a 48 hour range-finding test (nominal concentrations 1.0 and 100  mg/L), which is not fully reported, the definitive test was conducted at nominal test concentrations of 1.0, 3.2, 10, 32, and 100% of a 1.0 mg/L loading rate. A control group was also included.

In an effort to maintain the test concentrations and maximize exposure to CDBC, the test was conducted as a daily static renewal design (including week-ends).Ten replicate test vessels were prepared for each treatment and control group. A single juvenile Daphnia magna (<24 hours old) was added to each test vessel.

Each day during the renewal process, parental animals were transferred into freshly prepared test media. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on each day was recorded. At the end of the test, carapace lengths of all surviving parental Daphnia magna were measured microscopically.

Due to the low solubility and analytical method constraints it was concluded that it was not possible to determine reproducible analytical measurements for the test. The method used demonstrated that incurred samples had been exposed to the test substance but cannot be reliably quantified with the instrumentation currently available. Given the low solubility, solutions were prepared as saturated solutions and the analytical results, detailed in the below table, will only be reported as verification of dosing.

Mortality of Daphnia magna

No significant effects on the adult survival were observed at any test concentration when compared to the control treatments.

The NOEC value for adult survival was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on parent Daphnia magna mortality up to the water solubility limit of CDBC.

 

Daphnia magna Reproduction

No significant effects on the growth were observed at any test concentration as compared to the control treatments.

The NOEC value for growth was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on Daphnia magna reproduction up to the water solubility limit of CDBC.

 

Growth of Daphnia magna

No significant effects on the growth were observed at any test concentration as compared to the control treatments.

The NOEC value for growth was considered to be the 100% saturated solution of 1.0 mg/L nominal loading rate. It is concluded that no significant effect was observed on parent Daphnia magna growth up to the water solubility limit of CDBC.

No dose response and no significant effect were observed parent mortality or growth and reproduction. Therefore, it is concluded that CDBC has no long-term adverse effect on Daphnia magna up to its limit of water solubility.

Key value for chemical safety assessment

Additional information