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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:
13 February 2009 to 16 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All
- Sampling method: On days 0, 10 and 19 samples of the 'on' (freshly prepared) dilution water control and exposure concentrations were analysed for the concentration of the test material. Samples for the corresponding 'off' dilution water control and exposure concentrations (one replicate) were analysed on the following renewal day (days 3, 12 and 21). All 'off' samples were centrifuged prior to analysis. Triplicate analyses were performed on the nominal 1.0 mg/L exposure concentration on each sampling occasion.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock concentration of the test material with a nominal concentration of 100 mg/L was prepared. In each instance the concentrate was mixed by inversion and subjected to ultra-sonics for 10 minutes, which resulted in a clear, colourless solution. This newly prepared stock concentration was then used to prepared the 10 and 32 mg/L test solutions on each renewal occasion. This was achieved by the direct addition of the appropriate amounts of concentration to 1000 mL of dilution water in volumetric flasks. For the highest exposure treatment the concentration was used without any further dilution. The 0.32, 1.0 and 3.2 mg/L test solutions were prepared by serial dilution of the concentration by direct additions to 1000 mL of dilution water, also on each renewal occasion. All test solutions were observed to be clear and colourless.
- Controls: Dilution water (DWC)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age of parental stock (mean and range, SD): 32 ± 1 days old.
- Feeding during test: yes
- Food type: A mixed algal diet of C. vulgaris, strain CCAP211/12 and P. subcapitata, strain ATCC 2262.
- Amount: 0.06 mg C/day (equivalent to 0.12 mg C/daphnid/day).
- Frequency: Daily

ACCLIMATION
- Acclimation conditions (same as test or not): The parent animals had been maintained with a twice weekly renewal of reconstituted water medium since birth.
- Type and amount of food: Mixed algal diet.
- Feeding frequency: ad libitum.
- Health during acclimation (any mortality observed): The test organisms and the culture from which they were obtained showed no evidence of disease before the test period.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Culture conditions were such that the reproduction was by parthenogenesis. Daphnia < 24 hours old obtained from a single culture vessel were used for testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
248 to 258 mg/L as CaCO3
Test temperature:
19.4 to 20.2 °C
pH:
'on' solutions: 7.7 to 8.3
'off' solutions: 7.8 to 8.3
Dissolved oxygen:
'on' solutions: 8.6 to 9.2 mg/L
'off' solutions: 8.4 to 9.0 mg/L
Equivalent to a range of 93 to 101 % of the air saturation value
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Measured concentrations are presented in table 3 under the field "Any other information on results incl. tables". Across all exposure treatments, the mean measured concentrations of the 'on' test solutions ranged from 97 to 106 % of the nominal value, with the corresponding 'off' values ranging from 93 to 103 % of nominal. The analysis of the triplicate samples, taken from the nominal 1.0 mg/L solutions showed very good consistency.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers
- Type : Closed, covered with loose fitting glass lids
- Material, size, headspace, fill volume: Glass beakers containing 80 mL test solution (depth 50 mm)
- Renewal rate of test solution: The test solutions were prepared on the day of use and were renewed every Monday, Wednesday and Friday throughout the study. On each renewal day, new test solutions were dispensed into a second set of vessels and the surviving P0 (parent) generation were transferred to the new solutions.
- No. of organisms per vessel: 1 parent (P0) daphnid per vessel
- No. of vessels per concentration (replicates): 10 per exposure
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water medium used for testing (and maintenance of stock cultures) was Elendt's m4 D. magna medium. Details of water quality and preparation are presented in tables 1 and 2 respectively under the field 'Any other information on materials and methods incl. tables'.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light: 8 hours dark with 20 minute dawn and dusk transition periods.
- Light intensity: 7.2 µ Einsteins m^-2 s^-1

EFFECT PARAMETERS MEASURED:
Mortality of the P0 generation was recorded daily for each test vessel. Mortality was defined as animal being immobile or if there was no observed movement of appendages or post-abdomen within 15 seconds (after gentle agitation of the container if necessary). Observations were made at the same time for possible symptoms of toxicity. Observations were made daily from day 0 for the presence of live and dead offspring (F1 generation) in each vessel.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
Length
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
length
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: overall
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: overall
Details on results:
- Mortality of parent animals: Survival in the treated Daphnia was comparable to the control. Mortalities, presence of males and other visual observations in the P0 generation D. magna were recorded during the test. There was one mortality in the DWC after 16 days. This satisfies the guideline validity criteria of ≤20 % control mortality at the end of the test. Exposure concentration mortalities were observed in 32 mg/L (2 mortalities - days 13 and 14) and 100 mg/L (3 mortalities - days 9, 13 and 14).
- Body length and weight of parent animals: The lengths of the 32 mg/L and the 100 mg/L treatments were found to be significantly lower
than the DWC.
- Type and number of morphological abnormalities: A male was confirmed in both the 3.2 mg/L and 100 mg/L exposure concentrations. White spots were also noted in one daphnid in the 32 mg/L, which subsequently became a mortality. One D. magna in the 100 mg/L concentration was observed to be a male on day 7, however, this was re-evaluated and confirmed to be a female upon release of its first brood. One other D. magna in the 100 mg/L concentration was observed to have white spots on Day 15, but subsequently recovered. The incidence of males and white patches in the abdomen of P0 daphnids was considered to be unrelated to the test substance concentration since there was no pattern of increase in these observations with increasing test concentration.
- Time to first brood release or time to hatch: All P0 D. magna had released their first offspring by day 10
- Brood size The mean number of offspring produced in the control, per surviving parent, at the end of the test was 89, exceeding the minimum of 60 specified in the guideline. The residual standard deviation (coefficient of variation) around the mean number of living offspring per parent in the DWC was 13.8 %, and therefore below the maximum of 25% recommended in the guideline.
Reported statistics and error estimates:
Mortality: Survival in the exposure treatments was compared to the control using Fisher’s Exact Test. No significant reduction (p = < 0.05, bonferroni adjusted value p = 0.026) in survival in any exposure treatment was determined when compared to the control.

Length: Some of the data for length were found to be non-normally distributed and were therefore assessed for equality of variances between treatments using Levene’s test. Variances were found to be equal between treatments, therefore, a one-way ANOVA followed by a Dunnett’s test (two sided) was used to identify significant differences (p = 0.05) between the DWC and exposure treatments.

Reproduction: Prior to statistical analysis, an adjustment was made for mortality, by only including the P0 female D. magna surviving to the end of the study, and by excluding any males. The reproductive data were found to be non-normal and so were assessed for equality of variance with Levene’s test. Variances were found to be equal between treatments, therefore, a one-way ANOVA was used to identify significant differences (p = 0.05) between the DWC and exposure treatments.

Table 3: Analytical data

Nominal concentration of the test material (mg/L)

Exposure day

Measured concentration of test material (mg/L)

Percentage of nominal

On solution

Off solution

On solution

Off solution

Dilution water control

0

<LOQ

-

N/A

-

3

-

<LOQ

-

N/A

10

<LOQ

-

N/A

-

12

-

<LOQ

-

N/A

19

<LOQ

-

N/A

-

21

-

<LOQ

-

N/A

0.32

0

0.32

-

100

-

3

-

0.32

-

100

10

0.30

-

94

-

12

-

0.32

-

110

19

0.31

-

97

-

21

-

0.31

-

98

1

0

1.0

-

100

-

3

-

1.0

-

100

10

0.94

-

94

-

12

-

1.1

-

100

19

0.96

-

96

-

21

-

0.98

-

100

3.2

0

3.3

-

103

-

3

-

3.2

-

100

10

3.2

-

100

-

12

-

3.2

-

100

19

3.2

-

100

-

21

-

3.2

-

100

10

0

10

-

100

-

3

-

10

-

100

10

12

-

120

-

12

-

10

-

100

19

9.9

-

99

-

21

-

10

-

100

32

0

33

-

103

-

3

-

33

-

103

10

33

-

103

-

12

-

32

-

100

19

33

-

103

-

21

-

33

-

103

100

0

100

-

100

-

3

-

100

-

100

10

110

-

11 0

-

12

-

100

-

100

19

97

-

97

-

21

-

99

-

99

 

Analytical data continued

Nominal concentration of test material (mg/L)

Exposure day

Triplicate' values

1

0 on

1.0

1.0

1.0

3 off

1.0

1.0

1.0

10 on

0.93

0.95

0.95

12 off

0.97

1.2

1.0

19 on

0.95

0.95

0.97

21 off

0.97

0.99

0.99

 

Table 4: Adult length measurements

Nominal concentration of test material (mg/L)

Length in graticule scale divisions (day 21) per P0D. magna

Mean length

Standard deviation

Meana(mm)

1

2

3

4

5

6

7

8

9

10

Dilution water control

52

M

53

50

52

54

55

54

55

53

53

1.6

4.4

0.32

50

53

52

54

53

54

52

52

53

54

53

1.3

4.4

1.0

52

52

51

52

52

53

55

53

52

52

52

1.1

4.4

3.2

54

53

53

53

53

54

53

53

50

53

1.2

4.4

10

51

50

53

54

54

52

50

53

53

54

52

1.6

4.4

32

47

48

M

M

50

52

48

50

52

53

50b

2.2

4.2

100

49

49

46

45

M

M

47

46

M

47b

1.7

3.9

aLength in mm (1 mm = 12 scale divisions)

M = mortality

♂ observed male on day 7

bStatistically significant difference between treatment and dilution water control (p=0.005)

Table 5: Reproduction and mortality

Nominal concentration of test material (mg/L)

Total Number of Offspring per P0 D. magna

Mean per P0

Standard deviation

Total offspring

Mean offspring as % of dilution water control

(%) Mortality of P0 D. magna

1

2

3

4

5

6

7

8

9

10

Dilution water control

118

Ma

81

80

80

87

81

88

98

90

89.2

803

-

10

10

0.32

59

79

85

109

82

118

87

83

85

88

87.5

875

98

0

0

1

82

74

114

93

70

83

76

78

73

81

82.4

824

92

0

0

3.2

74

107

77

76

71

76

85

84

81

81.2

731

91

0

0

10

75

79

84

113

87

77

80

82

125

81

88.3

883

99

0

0

32

107

75

Mbef

Mc

80

78

113

78

76

81

86

688

96

20

20

100

49

55

39

41

Mc

Mb

50

31

Md

44.2

265*

50

30

30

M Mortality

aObserved to be dead on day 16

bObserved to be dead on day 13

cObserved to be dead on day 14

dObserved to be dead on day 9

♂ Observed to be male on day 7

* Statistically significant difference between treatment and dilution water control (p = 0.05)

eWhite patches observed in the abdomen (day 7)

fObserved to be transparent in appearance (day 9)

Table 6: Physical chemistry measurements

Nominal concentration of test material (mg/L)

Dissolved oxygen

pH

Temperature

Hardness as CaCO3

Minimum (mg/L)

Maximum (mg/L)

Minimum

Maximum

Minimum (°C)

Maximum (°C)

Minimum (mg/L)

Maximum (mg/L)

On

Off

On

Off

On

Off

On

Off

On

Off

On

Off

Dilution water control

8.8

8.4

9.0

9.0

7.8

8.0

8.3

8.3

19.4

20.2

248

248

254

254

100

8.6

8.6

9.2

9.0

7.7

7.8

8.3

8.3

248

254

253

258

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test, there were no statistically significant effects (p = 0.05) on P0 survival for any treatment, when compared to the control. There were statistically significant effects (p = 0.05) on P0 length for the 32 mg/L and the 100 mg/L concentrations, and on reproduction for the 100 mg/L concentration, when compared to the control. Taking all biological parameters into account the overall NOEC of the test material to Daphnia magna was determined to be 10 mg/L and the LOEC of the test material to Daphnia magna was determined to be 32 mg/L. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The effects of the test material to on the survival and reproduction on aquatic invertebrates was investigated in Daphnia magna in accordance with the standardised guidelines OECD 211. The Daphnia were exposed to the test material in a range of concentrations (0.32, 1.0, 3.2, 10, 32 and 100 mg/L under semi-static test conditions. The mean measured concentrations of the test substance were 97 -106 % ('on' values) and 93 -103 % ('off' values) of the nominal exposure concentrations; the results and conclusions were therefore based on nominal concentrations. There were no statistically significant effects (p <0.05) on survival for any treatment. The reproduction data showed that the offspring of the 100 mg/L treatment was found to be significantly lower when compared to the control. The lengths of the 32 mg/L and the 100 mg/L treatments were found to be significantly lower when compared to the control. The overall NOEC of test material was determined to be 10 mg/L. The LOEC of the test material was determined to be 32 mg/L.

Description of key information

21 day NOEC = 10 mg/L, 21 day LOEC = 32 mg/L, Daphnia magna, OECD 211, Hayfield 2009

21 day NOE = 8.88 mg/L, 21 day LOEC >8.88 mg/L, Daphnia magna, OECD 211, Zhen 2016

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
10 mg/L

Additional information

The effects of the test material to on the survival and reproduction on aquatic invertebrates was investigated in water flea (Daphnia magna) in accordance with the standardised guidelines OECD 211. The Daphnia were exposed to the test material in a range of concentrations (0.32, 1.0, 3.2, 10, 32 and 100 mg/L under semi-static test conditions. The mean measured concentrations of the test material were 97-106 % ('on' values) and 93 -103 % ('off' values) of the nominal exposure concentrations; the results and conclusions were therefore based on nominal concentrations. There were no statistically significant effects (p <0.05) on survival for any treatment. The reproduction data showed that the offspring of the 100 mg/L treatment were found to be significantly lower than the control. The lengths of the 32 mg/L and the 100 mg/L treatments were found to be significantly lower than the control. The overall NOEC of test material was determined to be 10 mg/L. The LOEC of the test material was determined to be 32 mg/L. The available data was considered to be complete and the result determined, 21 d NOEC value of 10 mg/L, was taken forward for risk assessment.

The results were confirmed in a second reproductive toxicity study with water flea, conducted to OECD TG 211 and under GLP, which was conducted as part of a notification in China. The study was conducted with semi-static renewal of medium every 72 hours. A range-finding study was performed at nominal test concentrations of 1.00 and 10.0 mg/L and the NOEC was determined to be 10.0 mg/L. Therefore, a limit test was performed at a nominal concentration of 10.0 mg/L (mean measured concentration: 8.88 mg/L) alongside a blank control. Based on mean measured concentrations, the 21-day NOEC for reproduction and survival was 8.88 mg/L (nominal concentration of 10.0 mg/L) and the 21-day LOEC for reproduction and survival was >8.88 mg/L (nominal concentration of > 10.0 mg/L).