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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 24, 2010 to April 15. 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Supplemented natural water was used for testing. The Bulk approach was applied.
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Supplemented natural water was used for testing. The Bulk approach was applied.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Samples of all the test concentrations and the stock were taken on Day 0, 2, 5, 14, 16, and 19. Some analysis was also conducted on Day 21.
- Analysis was undertaken immediately on samples if possible to quantify the concentration of the test substance in the test solutions. If this was not possible the samples were stored together with stability samples in the refrigerator until analysis. Aqueous samples were diluted 50:50 (v/v) with
leaching solution and filtered over a 0.45 µm Pall GHP membrane acrodisc syringe filter to remove the suspended solids and or algae before analysis.
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To prepare the stock solutions for every water renewal, on average 0.0051 g of test substance was weighed, and then was added directly to approximately 80 mL of the test media. This solution was sonicated for 2 min. A homogeneous solution resulted. This solution was allowed to cool whilst stirring. The resulting solution was then transferred to a 100 mL volumetric flask and the original vessel was rinsed with test media and the volumetric flask was made up to 100 mL also with test media. A slightly opaque but homogeneous stock was achieved.
- A fresh stock solution was prepared for each solution change. Test solutions were prepared by further dilution of the stock solution with the test medium to achieve the desired test concentrations. A geometric series of concentrations were used. The ratio between two consecutive concentrations was 3.2. Test vessels were filled directly from the intermediate stocks after a short stirring period. The solutions were renewed at least three times a wk during the test. One control containing only test medium was included in the test.


Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: Dr .U. Noack-Laboratorien Käthe-Paulus- Str. 1, D-31157 Sarstedt, Germany
- Age of parental stock (mean and range, SD): Less than 24 hours old and were obtained from parent animals reproducing parthenogenically. The animals used in the test were and aged between 2-4 weeks (having previously produced at least one brood before use). Offspring were
reference tested twice a year with potassium dichromate
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
All adults were inspected under magnification for abnormalities. Individuals were measured and dry weight was determined.
Hardness:
Ca – 40.0 mg/L
Mg – 1.43 mg/L
ºdH- 5.94ºdH (
Test temperature:
20.95 -21.90 °C.
pH:
7.1- 8.9
Dissolved oxygen:
The measured oxygen concentration did not fall below: 8.4mg O2/L.
Salinity:
Not reported
Nominal and measured concentrations:
- Nominal concentrations: 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L.
- Average recoveries of the test substance were >80%. Lower concentrations proved more difficult to recover and variation of results was higher.
Details on test conditions:
TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 50 mL beaker
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate:
- Food: Fed a diet of 0.1 - 0.2 mg of carbon per daphnid/d in the form of the algal strain Chlorella vulgari

OTHERS:
- The strain is cultured in the ECRA Environmental Chemistry laboratory and total organic carbon content has previously been measured and logged in the GLP system. Parent animals were checked for immobilisation on at least 6 occasions per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other abnormal observations were also recorded.
- Temperature and dissolved oxygen, were measured in the control and in the highest concentration both old and new solutions where parent
daphnids were still alive on Day 0, 5, 14, and 21. Continuous measurement of temperature in water next to the test apparatus was also conducted. The pH was measured more regularly on Day 0, 2, 5, 7, 9, 14 and 21.

Test medium
The test medium was prepared by mixing natural surface water with standard Elendt M4 media vitamins. The appropriate M4 stock was added to the natural surface water to give a concentration of 0.075 mg/L vitamin B1, 0.001 mg/L vitamin B12 and 0.00075 mg/L of Biotin in the final test media. This media was shown to provide sufficient nutrients to support good growth and reproduction of the test animals. As previously described natural surface water was used in this test in keeping with the bulk approach. Natural surface water was sampled from Heveadorp in the district of Renkum coordinates: 51º 58’N 5º48’E. The actual sample point is illustrated by an arrow and the text “hier monster genomen”. Natural surface water was frozen until use to preserve organic contents and reduce microbial activity. The dilution water was aerated before being used in the test. The air was water-saturated and purified by activated charcoal and a cotton filter.

Reference substance (positive control):
yes
Remarks:
Daphnia Culture referenced routinely with potassium dichromate.
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Length & Weight
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 1.15 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Adult Length
Results with reference substance (positive control):
- Reference tested as part of GLP maintenance twice per year with potassium Dichromate.
- Preliminary tests were conducted to establish definitive test concentrations and suitability of media to meet validity criteria was determined prior to testing.
Reported statistics and error estimates:
- The data was tested for normality using the Kolmogorov D test.
- Homogeneity of variance was confirmed by the Bartlett’s test.
- Analysis of variance using the Dunnetts test.
- The Fisher's exact test was also used for adult mortality endpoint. The calculations were conducted using Toxcalc 5.0.23 statistical software. The same statistical analysis was applied to Reproduction, length, weight and adult mortality endpoints where possible.

Results

Preliminary tests

No existing data was present on which to base the definitive test concentrations. For this reason an acute range finding was conducted in natural surface water following the relevant OECD Daphnia acute guideline and using the same stock preparation method as detailed. Five individuals were used per beaker in duplicate. The range finding results suggest that acute effect levels appear between 0.1 and 1 mg/L. As some mortality is desired in the highest concentration of the definitive chronic test 0.5 mg/L was chosen as the highest concentration. The largest factor permitted by the guidelines was applied to test a range as broad as possible to increase the chance of reaching a NOEC concentration in the definitive test.

Water quality

The observed temperature range which was continuously recorded during the test was: 20.95 -21.90°C. The measured oxygen concentration did not fall below: 8.4mg O2/L. The monitored water hardness in the test water batch was: 106 mg/L as CaCO3. (ºdH was measured and converted using a factor of 17.9 to convert to mg CaCO3/L) Light intensity was measured at the start at the end of the test on the laboratory table and was found to be 9.9 & 10.9 µmol/m2/s respectively. Nominal concentrations tested were 0.004, 0.015, 0.048, 0.15 and 0.5 mg/L. The highest 2 concentrations and the stock solution were analytically confirmed at 80 % of the nominals or greater. The remaining concentrations were detectable at fluctuating levels due to test substance binding to suspended matter.

Analytical results

The test concentrations 0.15, and 0.5 mg/L and the stock solutions were all measured at levels greater than 80% of the nominal concentrations. The 0.048 mg/L replicate fluctuated at around the 80 % recovery mark but fell slightly below this on a few occasions. The lower concentrations proved to be less stable and measured in some cases above the 80% level and in some cases considerably lower. It can be seen that at the lower concentrations the reproducibility of the analytical measurements is lower and considerably more variable than the higher concentrations. This is to be expected when measuring at such low levels with substances that adhere strongly to suspended matter. Based on the stock values, and when considering the overall analytical picture as well as the nominal amount of stock added to the replicates it can be concluded that with the exception of a few outliers (that will be discussed later) that the correct amount of test substance was added to the test vessels. Recovery levels are also high in general > 80%. The use of nominal concentrations for endpoint calculations is therefore justified. The results highlighted in red were considered to be outliers. The unexpectedly high recoveries in the 0.015 and 0.004 mg/L are caused by the lower repeatability and accuracy of the analytical method at lower concentrations. Only a slight fluctuation in the measured concentrations at 4 µg/L strongly influences the recoveries in comparison to the nominal values. Binding to suspended solids can also also result in an all or nothing analytical measurements at such low concentrations. Considering the stock analysis and the volumes added to these replicates from the stock the nominal concentrations of these results are theoretically correct but are at the limit of the analytical capability and carry therefore a greater error factor. The higher concentrations offer more stable, reproducible analytical measurements. A false measurement was also recorded in the stock measurement on day 2. This result can only be explained by the fact that the stock was sampled to early and not given enough time to stir. Such a high recovery is not possible when considering the amount of material that was added. It can be seen from the test concentrations that after homogenisation this had no negative effects on the test concentrations.

Other analytical results

Extraction of the test chemical from glass was validated in an algae test for the same test substance so was not revalidated as a part of this study. Measurements of the amount of test chemical adhering to the glass at 0.048 mg/L were conducted. 3.1% of the nominal concentration was recovered. This is negligible and glass binding will not have significantly affected the exposure. The remaining percentage of the same concentration in the water phase was 16.3 mg/L after 48 hours. This substance is not considered significantly biodegradable in the time between solution refreshments. The remainder of the test substance can be assumed to be bound to suspended solids or algae. Exposure was therefore satisfactory in keeping with the bulk approach.

Parent animal mortality

One adult daphnid died during the test period in the control. This is an acceptable mortality according to the appropriate OECD guideline. No adult daphnids died at 0.004 mg/L, 1 adult daphnid died at 0.015 mg/L, 1 adult died at 0.048 mg/L, 0 daphnids died at 0.15 mg/L and 2 daphnids died at 0.5 mg/L. A normal dose response effect was not observed for adult mortality. No significant effect in comparison to the control was detected using the Fishers exact test. The NOEC was therefore 0.5 mg/L and the LOEC >0.5 mg/L. The addition of the algae to the test replicates mitigated the effects of the test substance significantly with respect to the range finding test.

Coefficient of variation of control fecundity

The number of juvenile daphnids per replicate in each concentration was recorded. The recommended guideline criteria for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal surviving at the end of the test) was achieved. The mean number of neonates per surviving parent per replicate was also displayed. The total and the individual number of neonates per replicate were depicted.

Statistical results

The data on reproduction was tested for normality using the Kolmogorov D test and was found to be normally distributed. Homogeneity of variance was confirmed by the Bartlett’s test. Analysis of variance using the Dunetts test was performed on the number of living neonates per parent alive at the end of the test. Calculations were conducted using Toxcalc 5.0.23 statistical software. A NOEC for reproduction was calculated as 0.5 mg/L. A LOEC for reproduction was calculated as > 0.5 mg/L. An Ec10 for reproduction could not be calculated as a normal dose response effect was not observed in the data. Data based on adult length was also statistically analysed using the same approach as detailed above for reproduction. The calculations are presented in figure 4. A NOEC of 0.15 mg/L and a LOEC of 0.5 mg/L were calculated for the length endpoint. An Ec10 value for length was estimated without confidence limits as 1.15 mg/L using a probit plot. Weight data was also analysed in the same manner, normality of the data could not be confirmed most likely due to limited replicates but equal variances were confirmed for the data. The Dunetts test confirmed the data already observed for the length endpoint. A NOEC of 0.15 mg/L and a LOEC of 0.5 mg/L were calculated. An Ec10 for weight could not be calculated. A Fishers exact test was conducted on the adult mortality data using the same statistical software. A NOEC of 0.5 mg/L and LOEC of >0.5 mg/L were calculated.

Any other biological effects observed

Reduced feeding was clearly visible in the 0.5 mg/L replicate. This supports the statistical data from the length and weight endpoints demonstrating this concentration as a clear LOEC. No other biological abnormalities or effects were observed.

Conclusion

Based on adult mortality and reproduction no significant effects were observed. For length and weight endpoints statistically significant results were observed in the 0.5 mg/L test concentration making length and weight the most sensitive endpoints. The NOEC for adult mortality and reproduction was therefore 0.5 mg/L and the LOEC was >0.5 mg/L. The NOEC based on length and weight was 0.15 mg/L and the LOEC was 0.5 mg/L. The only Ec10 that could be calculated was for the length endpoint at 1.15 mg/L. Analysis has shown recovery in the stock and in the highest 2 concentrations and stock to be at least 80% of the nominal values. The lower concentrations could not be as accurately detected as in the higher concentrations. Recoveries as a percentage of the nominal concentrations fluctuated and fell below 80 % of the nominal values. It can be concluded however that the correct amount of test substance was added to the test vessels. This is reinforced by the amount of test chemical weighed into the stock, the analysis of the stock and the highest 2 concentrations and by the fact that the correct volumes of test substance were transferred from this stock to make the test concentrations. Due to the test substance binding strongly to suspended solids and the low detection limit required accuracy of analysis at low concentrations was limited and some outlying results were measured. Characteristics inherent to the test substance are acceptable as part of the bulk approach and would occur in the environment to a greater extent. Additional analysis demonstrated that the substance did not bind significantly to glass. The quality criteria set for the analytical method were met during measurement of the reported analytical results and the analytical method was concluded to be accurate at the time of analysis. In conclusion the test organisms were exposed to the test chemical in keeping with the bulk approach as far as was possible and the use of nominal concentrations for endpoint calculations was justified.

Amendments to and deviations from the study plan

(a) Amendments to the study plan

The study plan was amended due to a minor malfunction of the analytical apparatus. Due to a signal increase during long analytical runs calibration curves were analyzed every 6 samples to correct for this and ensure accurate analysis. The measurement of the apparatus was not affected.

(b) Deviations from the study plan

Water characterization measurements ammonium, total phosphate nitrate and total hardness were conducted as part of a GLP test but do not claim GLP compliance as the Dr Lange test system was not independently validated. Nominal concentrations were used for the calculation of effect levels in keeping with the bulk approach despite some recoveries falling below 80% of the nominal concentrations.

Quality criteria

The following quality criteria were met:

- 20% mortality occurred in the control group over the test period.

- The average number of juveniles per parent in the control was 142 after 21 days.

Natural surface water parameters

 

Parameter

Comment

Location

Heveadorp

Sample Date

17-3-10

Conditions when sampled

Windy ± 15ºc

Colour

clear- light yellow

Conductivity

313 µs/cm

NPOC

2.438 mg/L

TSS

18.2mg/L

Ammonium

0.041mg/L approx

Nitrate

5.79 mg/L

Total phosphate

0.083 mg/L

Total hardness

Ca – 40.0 mg/L

Mg – 1.43 mg/L

ºdH- 5.94ºdH (<LOD)

pH(with added Nutrients)

8.0

Analytical recovery

 Concentration

T=0 (%)

T=2d (%)

T=5d (%)

T=14d (%)

Control

--

--

--

--

0.004 mg/L

78.4

103.9

64.6

210.0

0.015 mg/L

80.4

95.1

73.0

110.7

0.048 mg/L

84.7

92.2

76.3

72.1

0.15 mg/L

81.9

86.1

79.5

92.9

0.5 mg/L

80.9

85.9

79.2

86.0

Test Stock

91.8

3267.9

83.1

107.4

  Concentration

T=16d (%)

T=19d (%)

T=21d (%)

Glass Extraction (%)

Control

--

--

 

 

0.004 mg/L

215.0

0.0

 

 

0.015 mg/L

82.7

46.0

 

 

0.048 mg/L

94.8

74.8

16.3

3.1

0.15 mg/L

85.8

87.7

 

 

0.5 mg/L

86.9

92.2

 

 

Test Stock

93.7

100.4

 

 

Some anomolous were results observed these are explained and justified in the report

Number of juvenile daphnids per parent alive at the end of the test at each test concentration, mean number of neonates per concentration and coefficient of variance

Concentration (mg/L)

Rep no.

0

0.004

0.015

0.048

0.15

0.5

1

184

137

135

133

144

121

2

117

130

137

134

159

119

3

147

136

147

149

162

133

4

D

138

153

142

159

157

5

141

159

D

157

155

151

6

151

127

130

132

166

D

7

143

165

144

119

160

135

8

148

149

120

D

156

D

9

107

128

143

126

165

112

z10

140

149

129

163

167

153

Mean

142

141.8

1.375.556

1.394.444

159.3

135.125

St. dev.

216.852.484

1.315.548

1.027.267

1.451.819

6.733.828

1.710.837

CV (%)

152.713.017

927.749

7.468.016

1.041.145

4.227.136

1.266.115

Total

1278

1418

1238

1255

1593

1081

Length of adult daphnids in units

 Daphnids No.

control

0.004mg/L

0.015 mg/L

0.048 mg/L

0.15mg/L

0.5mg/L

1

5.2

5.2

4.8

4.9

5.3

4.6

2

4.9

4.8

4.9

4.9

4.9

4.6

3

4.8

5.2

5.1

4.8

5.3

44

4

-

5.2

5.0

5.0

5.1

4.9

5

5.1

5.0

-

4.8

5.2

4.8

6

5.0

5.0

5.4

5.1

5.2

-

7

5.1

5.0

5.0

5.1

5.0

4.8

8

5.1

5.1

5.0

-

5.1

-

9

5.1

5.1

5.0

5.1

5.1

4.7

10

5.4

5.0

4.9

5.0

4.9

4.9

* 1 unit = 1mm

Weight of adult daphnids

Conc.( mg/L)

replicate

 

Weight before

No. of Daphnids’s

Total weight (g)

Weight per Daphnids (mg)

control

I

2.1782

4

2.1831

1.23

II

2.1889

5

2.1967

1.56

0.004mg/L

I

2.1953

5

2.2011

1.16

II

2.1929

5

2.1986

1.14

0.015 mg/L

I

2.1980

4

2.2024

1.10

II

2.1930

5

2.1989

1.18

0.048. mg/L

I

2.1793

5

2.1853

1.2

II

2.1544

4

2.1589

1.13

0.15mg/L

I

2.1498

5

2.1552

1.08

II

2.1485

5

2.1542

1.14

0.5mg/L

I

2.1497

4

2.1536

0.98

II

2.1438

4

2.1481

1.08

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively.


Executive summary:

A study was conducted to determine the long-term toxicity to Daphina magna of the test substance, C12-18 DAQ (99.6% active), according to the OECD guideline 211, in compliance with GLP.The test was

carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC) and Total Suspended Solids (TSS) content, allowing a more environmentally realistic determination of the effects of the test chemical to be made.The test organisms were exposed to test substance at nominal concentrations of 0, 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L for a period of 21d. During the test organisms were exposed to the test chemical in keeping with the bulk approach as far as was possible and the use of nominal concentrations for endpoint calculations was justified. Based on the observed results the NOEC for the test substance based on reproduction, mortality, adult length and weight were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively. The LOEC based on reproduction and adult mortality was therefore greater than >0.5 mg/L, whereas LOEC based adult length and weight was 0.5 mg/L. The EC10 based on length was calculated as 1.15 mg/L and confidence limits could not be calculated. Mortality in the control did not exceed 20% during the test period. The average number of juveniles per surviving parent in the control was 142. Under the study conditions, the 21 d NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively (Kean M, 2010).

Description of key information

Based on the study result, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.15 mg/L

Additional information

Study 1: A new study with the test substance according to OECD Guideline 211 is ongoing. Results will be added upon completion.

Study 2: A study was conducted to determine the long-term toxicity to Daphina magna of the test substance, C12-18 DAQ (99.6% active), according to the OECD guideline 211, in compliance with GLP.The test was

carried out according to the bulk approach using enriched natural surface water with a lowDissolved Organic Carbon (DOC) and Total Suspended Solids (TSS) content, allowing a moreenvironmentally realistic determination of the effects of the test chemical to be made.The test organisms were exposed to test substance at nominal concentrations of 0, 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L for a period of 21d. During the test organisms were exposed to the test chemical in keeping with the bulk approach as far as was possible and the use of nominal concentrations for endpoint calculations was justified. Based on the observed results the NOEC for the test substance based on reproduction, mortality, adult length and weight were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively. The LOEC based on reproduction and adult mortality was therefore greater than >0.5 mg/L, whereas LOEC based adult length and weight was 0.5 mg/L. The EC10 based on length was calculated as 1.15 mg/L and confidence limits could not be calculated. Mortality in the control did not exceed 20% during the test period. The average number of juveniles per surviving parent in the control was 142. Under the study conditions, the 21 d NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively (Kean M, 2010).