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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:
From Jan 19th to Feb 10th 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Temperature was outside the range stated in the guideline (17.8 to 21.3 °C instead of 18.0 -22.0 °C). As no immobility in the blank control occurred during the test and all validity criteria were fulfilled, this deviation was stated as uncritical
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Sample Preparation
Sample Preparation concentration 22 mg/L
After adding 6 g of NaCl, 50 mL of a solution in M4 medium was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20-50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconstituted using 5 mL of ethyl acetate.
An aliquot (2 mL) of the sample was added to 10 mL volumetric flask, 1 mL of 0.1% phe-nolphthalein in methanol solution; 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask was filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Twofold enrichment was achieved during the sample preparation.

Sample Preparation concentration 10 mg/L
After adding 6 g of NaCl, 100 mL of a solution in M4 medium was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20 - 50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconstituted using 5 mL of ethyl acetate.
An aliquot (2 mL) of the sample was added to 10 mL volumetric flask, 1 mL of 0.1% phe-nolphthalein in methanol solution; 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask was filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Fourfold enrichment was achieved during the sample preparation.

Sample Preparation concentration 4.6 mg/L
After adding 6 g of NaCl, 100 mL of a solution in M4 medium was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20 - 50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconstituted using 5 mL of ethyl acetate.
An aliquot (4 mL) of the sample was added to 10 mL volumetric flask, 1 mL of 0.1% phe-nolphthalein in methanol solution; 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask was filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Eightfold enrichment was achieved during the sample preparation.

Sample Preparation concentration 2.2 and 1.0 mg/L and the blank control
After adding 6 g of NaCl, 100 mL of a solution in M4 medium was extracted two times with the solvent ethyl acetate (15 and 10 mL), the organic phase was collected into a 20 - 50 mL pear shaped flask, after drying with Na2SO4 and evaporated to dryness. The residue was then reconstituted using 5 mL of ethyl acetate.
The sample was added to 10 mL volumetric flask, 1 mL of 0.1% phenolphthalein in meth-anol solution; 1 mL of 0.1 M phenyltrimethylammonium hydroxide was added and the flask was filled up to 10 mL with ethyl acetate. Internal standard solution can be added to the test solution before filling the flask. Tenfold enrichment was achieved during the sample preparation.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
Species Daphnia magna
Variety STRAUS
Strain Berlin
Arrival of Strain 27. Sep. 2007
Sex female
Age between 0 and 24 hours
The animals which were used in the test did not belong to the first brood of the respective parent animals.

Animal Husbandry
Daphnia magna is bred in the LAUS GmbH throughout the year. The animals were origi-nally obtained from the Umweltbundesamt in Berlin and are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical. The keeping is performed similar to the method described in EN ISO 6341, following SOP 115 002 01 (“Zucht und Hälterung von Daphnia magna STRAUS“), edition 12, adopted 02. Feb. 2015.
Vessels glass beakers, nominal volume 2 litres
Medium M4-Medium (recipe of ELENDT)
Food green alga (Desmodesmus subspicatus)
Medium renewal twice a week
Photo period 16/8 hours, using neon tubes
Temperature 20 ±2 °C
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
267 - 356 mg CaCO3/L
Test temperature:
17.8 - 21.3 °C
pH:
Experiment 1: 7.4 - 8.5
Experiment 2: 7.3 - 8.1
Dissolved oxygen:
Experiment 1: 8.5 - 10.6
Experiment 2: 7.9 - 9.8
Nominal and measured concentrations:
Nominal Geometric mean measured
Concentrati Concentration
(mg/L) (mg/L)

1.0 0.33
2.2 0.54
4.6 0.95
10 2.01
22 4.83
Details on test conditions:
Duration 21 days
Loading 80 mL/animal, animals were kept individually
Replicates 10 replicates/treatment
Treatments 1.0 / 2.2 / 4.6 / 10 / 22 mg/L nominal concentration
Blank Control M4-medium
Lighting 1000 Lux
Aeration none
Feeding 0.20 mg organic carbon/animal/day, the amount of carbon is based on photometric measurement (linear correlation between absorption at 440 nm and measured DOC con-centration)
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 4.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 4.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 4.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation

Findings and Result

 Reproduction

In the following table, the summary of the reproduction is given.

Replicate

Blank Control

1.0 mg/L

2.2 mg/L

4.6 mg/L

10 mg/L

22 mg/L

1

75

115

130

78

114

109

2

99

M

M

78

112

134

3

98

M

M

M

88

116

4

94

69

91

75

90

104

5

84

89

120

69

129

84

6

117

112

79

74

80

103

7

79

121

93

70

91

123

8

93

130

104

M

92

101

9

89

105

90

117

119

103

10

120

101

75

94

120

M

Mean

95

105

98

82

104

109

SD

15

19

19

16

17

14

RSD

15.53

18.30

19.61

19.73

16.45

13.25

SD = standard deviation

RSD = relative standard deviation = coefficient of variation

M = mortality of parent animal

 

Summary of Fecundity

In the following table, the total living offspring per treatment and the corresponding living offspring per surviving parent animal and the inhibition in comparison with the control is stated:

Concentration in mg/L

Total Offspring

Surviving Parent Animals

Mean Offspring per Surviving Parent Animal

Inhibition of Reproduction in %

Blank Control

948

10

95

0

1.0

842

8

105

-11

2.2

782

8

98

-3

4.6

655

8

82

14

10

1035

10

104

-9

22

977

9

109

-15

Negative inhibition values indicate a stimulation of fecundity compared to the control.

Validity criteria fulfilled:
yes
Conclusions:
No toxicity observed at any concentration tested.
NOEC 21d Daphnia > 4.83 mg/L, based on geometric mean of measured highest concentration (nominal: 22 mg/L)
Executive summary:

One valid experiment was performed.

10 Daphnia were exposed to the test item for each treatment and blank control for 21 days in a semi-static test system.

The study was performed using five concentrations ranging from 1.0 to 22 mg/L. The water-accommodated fraction (WAF) was prepared for the test.

The offspring of the animals was counted each working day. Reproduction of the daphnia in the highest treatment was higher than in the blank control.

The content of the test item in the test solutions was determined using GC-MS determination. The analysis of the test item concentrations in the treatments showed a low correlation with the nominal concentrations because of the limited solubility in test medium. Therefore, the determination of the results was based on the geometric mean of the measured concentrations.

According to the OECD guideline, the offspring of dead animals must be taken into account if the death is caused by toxicity of the test item. Only inadvertent and/or accidental parental mortality should be excluded from evaluation. Because no toxicity could be observed, the offspring of deceased parent animals was excluded from the calculation of total offspring per treatment.

 

The following results were determined for the test item DIACID 1550:

 

Parameter

Measured Concentration

NOEC

≥4.83 mg/L

LOEC

> 4.83 mg/L

EC50

> 4.83 mg/L

Description of key information

NOEC 21 d Daphnia magna (OECD 211): >5.3 mg/L 

Key value for chemical safety assessment

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

Additional information