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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:
09.05.2017 - 16.06.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Adopted: October 2, 2012
Deviations:
yes
Remarks:
see Any other information
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Remarks:
Only the concentrations of metals in the highest loading rate 10 mg·L-1 were analysed once a week (in the fresh and old solutions) in the preliminary test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Because the test substance is a multi-component poorly water-soluble substance the water-accommodated fractions (WAFs) were prepared. Preparation of WAFs was carried out according to Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD Environmental Health and Safety Publication, Series on Testing and Assesment No. 23, Paris 2000, Section 3.11.)
The WAFs were prepared by adding measured amounts of the test substance to the M4 medium and then were stirred for suitable period. After period of stirring the WAFs were filtered through 0.45 μm filter and these solutions were used for testing.
The suitable period for achieving equilibrated concentration of dissolved and dispersed components in aqueous phase was determined in preliminary experiment. For preliminary experiment the WSFs were prepared separately as follows: the suspension of 10 mg of the test substance in 1 000 mL of M4 medium was stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, subsequently filtered through 0.45 μm filter. During this experiment the concentrations of metals (Na, Ca, Fe, K, Mg, Mn, P, Si) in resulted WSFs were determined by atomic spectrometry method.
On the basis of the measured concentrations of metals, the period of 3 hours stirring was chosen as appropriate for the pretreatment of the test substance in the test. Non dissolved material was presented in the vessels after stirring. The non dissolved material present in WAFs has a potential to exert physical effects as encapsulation/entrapment of daphnids which are unrelated to toxicity. For this reason the all tested WAFs were filtered through 0.45 μm filter after stirring period to get water-soluble fractions for the exposure experiment.




Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Daphnia magna Straus
- Source: aperiodic parthenogenesis from own laboratory breeding
The grow-up gravid females are separated into M4 medium. Young daphnia born in 24 hours are segregated by observation under a microscope. Only the females are transferred to the test containers.
- Feeding during test:
daily (out of 27.05., 28.05., 03.06., 04.06., 10.06., 11.06.), the mixture of two algae (Desmodesmus subspicatus and Raphidocelis subcapitata 1:1), ration levels 0.5 mg C/daphnia/day at all times
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
2.47 – 2.65 mmol Ca+Mg /L
Test temperature:
20 – 21°C; in general 20 ± 2°C, with a variation of ± 2°C at each individual test
pH:
7.6 – 8.3
Dissolved oxygen:
6.8 – 8.2
Salinity:
not applicable
Conductivity:
1.46 – 1.87 µS·cm-1
Nominal and measured concentrations:
Preliminary test: WSFs 10, 5, 1 mg·L-1
On the basis of the preliminary test results and in congruence with study plan, no further testing was needed.
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 ml
- Aeration: without
- Renewal rate of test solution (frequency/flow rate): 3× per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.5 mg C/organism/day
- Volume of the mixture of algae for feeding: 172 µL/100 mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Test medium: M4
- Conductivity: 1.46 – 1.87 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: 1520 – 1525 lx (not exceeding 1250 – 1670 lx)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The following data were recorded during the test: numbers of living offspring by each parent animal, presence of dead offspring and mortality parent animals. The total number of offspring per parent animal was calculated for each test vessel. If in any test vessel the parent animal died during the test, then the test vessel was excluded from the analysis.
The mean reproductive output for each loading rate and control were calculated. Reduction of reproductive output was expressed in %.
- Mortality of control: 1 parent animal (17th day); 0 offspring


Results with reference substance (positive control):
a) Reference test
The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference substance, potassium dichromate. The results of the verification test with K2Cr2O7 are the following:
21 days – EC50 = 0.39 mg/L (95% confidence limit: 0.19 – 0.81 mg/L)

b) Data in the literature
HERMENS, JOOP, et al. Joint effects of a mixture of 14 chemicals on mortality and inhibition of reproduction of Daphnia magna. Aquatic Toxicology, Volume 5, Issue 4, November 1984, Pages 315-322.
21 days – EC50 = 0.27 mg/L

c) Method validity evaluation
The reduction in reproductive output of the test organism by the reference substance
(21d – EC50) obtained from our reference test is conformable to the values stated in the literature (Hermens et al., 1984).

Number of offspring

10 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

66

0

0

2

54

0

0

3

65

0

0

4

67

0

0

5

60

0

0

6

70

0

0

7

61

0

0

8

58

0

0

9

51

0

0

10*

16

0

1 (17th day)

Mean number of living offspring

61.3

Total number of dead parent animals

1

5 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

57

0

0

2

69

0

0

3

72

0

0

4

68

0

0

5

62

0

0

6

52

0

0

7*

0

0

1 (14th day)

8

70

0

0

9

63

0

0

10

61

0

0

Mean number of living offspring

63.8

Total number of dead parent animals

1

1 mg/L

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

74

0

0

2

63

0

0

3

61

0

0

4

54

0

0

5

59

0

0

6

66

0

0

7

62

0

0

8

52

0

0

9

68

0

0

10

61

0

0

Mean number of living offspring

62.0

Total number of dead parent animals

0

control (M4)

Vessel No.

(one parent animal/vessel)

Living offspring

Dead offspring

Dead parent animals

(day of death)

1

59

0

0

2

63

0

0

3

60

0

0

4

58

0

0

 5*

20

0

1 (17th day)

6

65

0

0

7

62

0

0

8

50

0

0

9

68

0

0

10

63

0

0

Mean number of living offspring

60.9

Total number of dead parent animals

1

* - not included in calculation of means

Validity criteria fulfilled:
yes
Remarks:
see Overall remarks
Conclusions:
The highest observed reduction in reproductive output of Daphnia was 0 % in the preliminary test. Therefore exact values of EL50 could not be calculated and the values of EL are given in the form of a range. The loading rates of the test substance were used for all evaluations and results.
Preliminary test showed that it could be reasonable to expect that estimated EL50 will be higher than 10 mg/L:
21 days–EL50 > 10 mg/L (loading rate)
In congruence with study plan, when the assumed value of EL50 is higher than 10 mg/L, no further testing is needed and the result obtained in preliminary test presents the result of whole study.
Executive summary:

The test substance, SDA Product, was tested in reproduction test on Daphnia magna. The test was performed according OECD Test Guideline No. 211 - Daphnia magna Reproduction Test. Adopted: October 2, 2012.

Since the test substance is a multi-component poorly water-soluble substance the water-accommodated fractions (WAFs) were prepared by adding measured amounts of the test substance to the M4 medium and then were stirred for a period of 3 hours. After the period of stirring the WAFs were filtered through 0.45 μm filter and these water-soluble fractions (WSFs) were used for testing.

The preliminary test was performed in a range of the test substance loading rates 0.1 – 10 mg/L. The test was performed as semi-static, frequency of the test solutions renewal was three times a week. The highest calculated reduction in reproductive output was 0 %, the exact values of EL50 could not be calculated on the basis of data acquired. Test result is given in the form of a range. In congruence with study plan, when the assumed value of EL50 is higher than 10 mg/L, no further testing is needed and the result obtained in preliminary test presents the result of whole study.

The study was performed without the analytical determination of the test substance concentrations. Only the concentrations of metals in the loading rate 100 mg·L-1 were analysed in the preliminary experiment. Original purpose of metals determination was the choice of suitable period of stirring in the course of WSFs preparation.

For all evaluations and results the loading rates of the test substance were used.

Test results, based on nominal loading rate: 21 days–EL50 > 10 mg/L (loading rate).

Description of key information

The highest observed reduction in reproductive output of Daphnia was 0 % in the preliminary test. Therefore exact values of EL50 could not be calculated and the values of EL are given in the form of a range. The loading rates of the test substance were used for all evaluations and results.

Preliminary test showed that it could be reasonable to expect that estimated EL50 will be higher than 10 mg/L:

21 days–EL50 > 10 mg/L (loading rate)

In congruence with study plan, when the assumed value of EL50 is higher than 10 mg/L, no further testing is needed and the result obtained in preliminary test presents the result of whole study.

Key value for chemical safety assessment

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

Additional information