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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:
27 April - 18 May 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
No phys-chem data, analytical method validation is lacking. Actual conc measurements day 3-5 unreliable, however, the final conclusion is not influenced and therefore is valid. Use of vehicle requires caution to extrapolate end-point to actual situation without vehicle. Identity and composition data given in appendix but batch no. not the same as the one of the supplier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
On seven test solution renewal occasions samples of each new test solution were analysed for the concentration of Aquapel 364. samples from the corresponding old test solutions (one replicate) were analysed on the following renewal day.
Vehicle:
yes
Details on test solutions:
Vehicle was actone.
The study was run with a dilution water and solvent control together with nominal
Aquapel 364 concentrations of 0.056, 0.1, 0.32, 0.56 and 1.0 mg/L

A primary stock suspension, prepared by dispersing 0.1 g of Aquapel 364 in 10 ml
acetone, was used to prepare the test solutions. These suspensions were prepared on each
test solution renewal occasion and were observed to be cloudy white homogeneous
suspensions.
On each test solution renewal day, the test solutions were prepared by the addition of
appropriate aliquots of the primary stock to dilution water. The dilution water control,
solvent control and test solutions were observed to be clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test species was the freshwater crustacean, Daphnia magna, obtained from continuous laboratory cultures.
The stock cultures of Daphnia were maintained in a reconstituted water medium, identical to the test dIlution water, at a temperature of 20 ± 1DC for a minimum of 3 weeks prior to the start of the study. The cultures were maintained in 2 L glass vessels with a working volume of 1.5 L. A photoperiod of 16 hours light8 hours dark, with 20 minute transition periods was provided. The Daphnia cultures were fed a defined diet of algae ChIorella vulgaris, strain CCAP 211/12 and a commercially available microencapsulated diet "Frippak Booster®". Culture conditions were such that the Daphnia reproduction was by diploid parthenogenesis. The Daphnia cultures were fed daily ad libitum depending on age and density of the culture.

Daphnia <24 hours old (first instar), obtained from a single culture vessel, were used for testing. The parent animals were 17 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium, since birth. The test organisms and the culture from which they were obtained showed no evidence of disease before the test period.

The test Daphnia were fed daily with cultured algae (Chlorella vulgaris), strain CCAP 211/12 and a commercially available microencapsulated diet "Frippak Booster®". The algal cultures were harvested and centrifuged and the algal cells resuspended in Daphnia culture medium (reconstituted water). The algal stocks were measured for cell density using a Coulter Counter Model ZB, stored in a refrigerator, in the dark, and used within 1 week. The algal stocks were also analysed for total organic carbon content. A suspension of "Frippak Booster®" was prepared every Monday, Wednesday and
Friday by dispersing 0.1 g in 100 ml of test dilution water and was stored in a refrigerator. Each test vessel received 3.0 X 10^7 algal cells and 57 ug of "Frippak Booster®" (as a suspension) per day.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
as CaCO3 210-218.7mg/L
Test temperature:
19.6-20.9 C
pH:
new solutions: 8.0 - 8.3
old solutions: 7.6 - 8.2
Dissolved oxygen:
new solutions: 8.8-9.4 mg/L
old solutions: 7.8-9.4
Nominal and measured concentrations:
nominal: 0.056, 0.10, 0.32, 0.56, 1.0 mg/L
mean measured new: 0.076, 0.11, 0.28, 0.49, 0.88 mg/L
mean measured old: <0.071, 0.12, 0.26, 0.44, 0.72 mg/L
Details on test conditions:
Borosilicate glass beakers were used as test vessels, with 10 replicates per exposure concentration, each containing 80 ml of test solution providing a depth of 47 mm. The beakers were covered with loose fitting lids.
The nominal test solution temperature was 20 ± 1 C and a photoperiod of 16 hours light: 8 hours dark with 20 minute transition periods. Light intensity was measured once during the study, and was taken at the surface of the test solutions. The test solutions were not aerated.
The positions of the grouped test treatments were randomly allocated within the test area.
At the start of the test, one Daphnia was randomly selected and added to each test vessel in sequence across the treatments. These Daphnia were termed the Po generation. Mortality of the Po generation was recorded daily for each test vessel. Mortality was defined as absence of any movement by the organism, when examined by eye, for a period of 15 seconds (after gentle agitation of the container if necessary). Observations were made at the same time for any other symptoms of toxicity. Observations were made daily from day 4 for the presence of live and dead offspring (termed F1 generation) in each vessel.
The test solutions were prepared on the day of use and were renewed every Monday, Wednesday and Friday. On each renewal day, new test solutions were dispensed to a second set of test vessels. The surviving Po generation were transferred to the new solutions, using a wide bore pipette, minimising transfer of medium. The old solutions were sampled, as appropriate, for physical and chemical analyses and the P1 generation removed from each vessel and counted. The numbers of live and dead P1 were recorded. The duration of the test was 21 days.
Daphnia was measured, using a microscope with an eyepiece graticule previously calibrated with a stage micrometer. The median lethal effect concentration (LC50) was defined as the concentration resulting in 50% mortality of the Daphnia in the time period specified.
The reproduction data for each individual Po generation daphnid were entered into electronic data files and analysed using statistical procedures. The data were tested for normality and if the distribution was normal, Bartlett's test for homogeneity of variance was applied. If the data had a normal distribution and the variance was homogeneous then the data were analysed using a parametric statistical procedure such as Dunnett's T test following analysis of variance. If the data were not normal or failed Bartlett's test for homogeneity of variance, then the data were analysed using a non-parametric statistical procedure such as Wilcoxon's Rank Sum Test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
An adjustment was made for mortality in the statistical analyses by only including Po Daphnia surviving to the end of the study.
In the dilution water and solvent controls and all nominal test concentrations up to and including 1.0 mg/L all Po Daphnia had released their first offspring on day 10 and had completed at least 4 broods by the end of the study.
Compared to the pooled dilution water and solvent controls there was no significant decrease (P=0.05) in the numbers of live offspring (F1 ) produced in any of the nominal test concentrations.
Therefore, the no observed effect concentration (NOEC) for reproduction was 0.8 mg/L and the lowest observed effect concentration (LOEC) was >0.8 mg/L. The EC50 was estimated to be >0.8 mg/L

Measurements at day 3 -5 from the new solutions are less compared to the old solutions.
The pH min-max range is higher than 1.5 and the maximum pH (9.4) exceeds the limit in the guideline of 9.
Possible contamination of samples durig preparation of samples for analysis.
Mean measured concentrations in new test solutions ranged from 88 to 136%, but individual data indicate a range 57-290%
Reported statistics and error estimates:
The reproduction data were not normally distributed, therefore, a non parametric statistical procedure, Wilcoxon's Rank Sum Test, was used to analyse the data.

Table 1: P0generation mortalities

Nominal [Aquapel 364] mg/L

Day of Test

Number dead per replicate

% dead

1

2

3

4

5

6

7

8

9

10

Water contr.

21

0

0

0

0

0

0

0

0

0

0

0

Solvent contr.

21

0

0

0

0

0

0

0

0

0

0

0

0.056

11

0

0

0

0

0

0

0

0

1

0

10

21

0

0

0

0

0

0

0

0

1

0

10

0.10

21

0

0

0

0

0

0

0

0

0

0

0

0.32

4

0

0

0

0

1

0

0

0

0

0

10

6

0

0

0

0

1

0

1

0

0

0

20

21

0

0

0

0

1

0

1

0

0

0

20

0.56

14

0

0

0

0

1

0

0

0

0

0

10

21

0

0

0

0

1

0

0

0

0

0

10

1.0

21

0

0

0

0

0

0

0

0

0

0

10

 

Table 2: Reproduction data

Replicate number

Total of Offspring per parent

Nominal concentration of Aquapel 364 mg/L

Water control

Solvent control

0.056

0.10

0.32

0.56

1.0

1

84

86

91

97

90

75

30

2

86

95

94

83

96

85

101

3

89

89

85

90

86

93

90

4

89

93

97

98

102

98

92

5

88

65

84

97

(0)

(47)

88

6

89

83

103

90

94

95

87

7

86

86

64

99

(0)

99

95

8

90

82

100

101

100

91

97

9

84

90

(33)

76

76

72

89

10

62

91

96

100

100

96

91

Mean

85

86

90*

94*

93*

88*

86*

SD

8.3

8.5

11.8

59

8.8

10.0

20.2

CV

9.8

9.9

13.1

6.3

9.5

11.4

23.5

Total

847

857

814

938

774

794

860

% dead

0

0

0

0

0

0

0

* = No significant decrease compared with pooled dilution water and solvent controls

() Values in parenthesis denote offspring from P0 Daphnids that had dies before day 21. these data were not included in the mean, SD and statistical analyses but were included in the total number of offspring.

 

Table 3: Adult length measurement

Replicate number

Length in graticule scale divisions (Days 21)

Nominal concentration of Aquapel 364 mg/L

Water control

Solvent control

0.056

0.10

0.32

0.56

1.0

1

55

54

55

54

55

55

55

2

55

57

57

57

56

55

52

3

51

56

55

55

55

57

55

4

53

55

56

55

55

55

55

5

55

59

53

55

-

-

55

6

50

56

55

55

55

51

55

7

55

50

54

56

-

56

54

8

55

60

55

55

56

51

60

9

55

55

-

56

60

57

57

10

54

55

55

55

56

55

55

Mean

54

55

55*

55*

56*

55*

55*

SD

1.9

3.1

1.2

0.8

1.7

2.0

2.0

Mean length

Length in mm (1mm = 12 divisions)

4.50

4.58

4.58

4.58

4.67

4.58

4.58

*= No significant decrease compared with pooled dilution water and solvent controls

- = mortality before day 21

Validity criteria fulfilled:
yes
Conclusions:
CoA does not match batch no. provided by supplier. No phys-chem data. Analyses day 3-5 are questionable, some contamination of samples, analytical method validation is lacking. Due to lack of effects wrt growth or reproduction, the data can still be used, however the use of vehicle implies caution as to extrapolate to normal conditions.

therefore valid
Executive summary:

The chronic toxicity of Aquapel 364 has been determined for Daphnia magna according to the OECD guideline (211). The test substance was solved using a vehicle (acetone) and the test was semi static. 10 daphnids were used per concentration level, each housed individually.

Concentrations tested ranged from nominally 0.056 - 1.0 mg/L, however actual concentrations varied from 57%-290% of nominal. Some mortality (up to 20%) was observed, but not dose-dependent. No significant effects were determined for reproduction or length. The overall no observed effect concentration was > = 0.8 mg/L and the overall lowest observed effect concentration was > 0.8 mg/L based on mean measured concentrations.

Description of key information

Key study is Brixham Environmental Laboratory (2001). OECD211, 21day daphnia magna. Vehicle (acetone) used. No statistical significant effects on reproduction 
and mortality at highest concentration tested (nominal 1 mg/L, mean measured 0.8 mg/L). Study reliable with restrictions. End-point for derivation of PNEC
should not be concluded on acetone assisted actually measured substance concentration. Under normal conditions acetone will not be present as solvent.
Based on mean measured concentration the NOEC is >= 0.8 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
0.8 mg/L

Additional information

No statistical significant effects on reproduction and mortality at highest concentration tested (nominal 1 mg/L, mean measured 0.8 mg/L). Study reliable with restrictions, identity and composition data given in appendix but batch no. not the same as the one of the supplier. No phys-chem data, analytical method validation is lacking, actual conc measurements day 3-5 unreliable. However, the final conclusion is not influenced and therefore is valid. Use of vehicle requires caution to extrapolate end-point to actual situation without vehicle.