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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:
2015-08-19 to 2015-09-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 211 with GLP statement. One deviation was observed. Indeed, according to the most recent OECD Guideline 211 (2012), the response variable number of living offspring produced per surviving parental animal has been supplemented with an additional response variable for Daphnia reproduction: “total number living offspring produced at the end of the test per parent daphnia at the start of the test (excluding from the analysis parental accidental and/or inadvertent mortality)” but this is not mentioned in the report. This deviation was not considered to have impacted on the integrity of the study. This study is considered reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
. The response variable for Daphnia reproduction “total number living offspring produced at the end of the test per parent daphnia at the start of the test" is not mentioned in the report.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling for the analytical monitoring: At the start of the exposure intervals (0 hours), sampling was carried out after preparation of the test concentrations. At the end of the exposure intervals (48 or 72 hours), samples were taken from additional replicates without daphnids and food algae. The additional replicates were incubated under test conditions until sampling. For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period.



Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

Preparation of the stock solution:
A stock solution (8.00 mg/L of the test item were weighed out) wasfreshly prepared with dilution water for each exposure interval.

Treatment:
The stock solution was stirred with a magnetic stirrer at approximately 1100 rpm for 1 hour at room temperature. The stock solution was used for preparation of further test concentrations and as highest test concentration.

Test concentrations:
5 test item concentrations in a geometric series with a separation factor of 2.5 were prepared by diluting the stock solution of 8.00 mg/L with the
dilution water and tested as follows: 0.205 - 0.512 - 1.28 - 3.20 - 8.00 mg/L.

Control:
10 replicates of dilution water without test item

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Renewal of the test solution: The test solutions were renewed 3 times per week (i.e. after 48 or test solutions 72 hours, on Monday, Wednesday and Friday). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by
pipette

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Less than 24 hours old daphnids from a healthy stock were used at the start of the exposure for the study. Juvenile daphnids were removed from the culture vessels 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the
exposure were used for the test. No first brood progeny was used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
none
Post exposure observation period:
Not observed
Hardness:
Total Hardness [mg/L] as CaCO3

Geometric
mean measured
of the test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2015-08-19 2015-08-21 2015-08-28 2015-08-31 2015-09-02 2015-09-04
6.91 166 163 171 166 165 167
Control 167 164 164 164 166 166



Test temperature:
Temperature [°C]

Geometric
mean measured
of the test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2015-08-19 2015-08-21 2015-08-28 2015-08-31 2015-09-02 2015-09-04
6.91 20.7 20.5 20.9 20.6 20.1 20.4
Control 20.0 20.7 20.3 20.5 20.6 20.5




pH:
pH-Values

Geometric
mean measured
of the test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2015-08-19 2015-08-21 2015-08-28 2015-08-31 2015-09-02 2015-09-04
6.91 7.98 7.67 7.71 7.74 7.98 7.89
Control 8.02 7.68 8.32 7.66 7.94 7.94




Dissolved oxygen:
Dissolved Oxygen Concentration [mg/L]

Geometric
mean measured
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2015-08-19 2015-08-21 2015-08-28 2015-08-31 2015-09-02 2015-09-04
6.91 8.98 10.3 8.66 9.65 9.16 9.04
Control 8.99 8.70 8.88 9.03 9.23 9.60




Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to "Any other information on materials and methods".

5 test item concentrations in a geometric series with a separation factor of 2.5 were prepared by diluting the stock solution of 8.00 mg/L with the dilution water and tested as follows: 0.205 - 0.512 - 1.28 - 3.20 - 8.00 mg/L. The geometric mean measured concentrations of the test substance are: 0.0432 - 0.0693 - 0.138 - 1.83 - 6.91 mg/L. Since the measured concentrations did not remain within ± 20 % of the nominal or initially measured concentrations, all effect values given are based on the geometric mean measured concentrations of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): loosely covered with a watch glasss
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal of the test solution: The test solutions were renewed 3 times per week (i.e. after 48 or 72 hours, on Monday, Wednesday and Friday).
For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette

- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 h light/dark cycle
- Light intensity (target): Max. 20 µEm-2 s-1 (1340 lx)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end






Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: < 0.0432 - 3.11 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.138 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
1.83 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
• The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 84 in the control group.
The reproductive output was statistically significant reduced at the concentration level of 1.83 mg/L (21.3 %) in comparison to the reproductive
output in the control (One Way Analysis of Variance, DUNNETT’S Method, P < 0.05). In the concentration levels of 0.0432 to 0.138 mg/L, the
reproductive output was comparable to the reproductive output in the control. In the concentration level of 6.91 mg/L, all daphnids died before
producing juveniles and therefore the reduction of the reproductive output was set to 100 %.
The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 1.02 mg/L
(95 % confidence limits: < 0.0432 - 3.11 mg/L). The EC50 for the reduction of the reproductive output was calculated accordingly to be 3.70 mg/L (95 % confidence limits: 2.12 - 5.37 mg/L).

• Based on the significant reduction of the reproductive output at the concentration level of 1.83 mg/L, the No Observed Effect Concentration
(NOEC) after 21 days was assessed at 0.138 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 1.83 mg/L.

• The coefficient of variation of the number of living offspring produced per surviving parental daphnid was 6 % in the control.
At the concentration levels of 0.0432 to 1.83 mg/L, the coefficients of variation were in the range of 7 to 10 % . Generally in a well-run test,
the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %.

• The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of natural increase determined for the surviving daphnids at the
concentration level of 1.83 mg/L was statistically significant in comparison to the IR at the control (One Way Analysis of Variance, DUNNETT’S
Method, P < 0.05). At the concentration levels of 0.0432 to 0.138 mg/L, the IR were comparable to the IR at the control. Due to 100 % adult
mortality, no IR could be calculated at the concentration level of 6.91 mg/L.

• At the end of the test after 21 days, the number of broods with living juveniles produced by the surviving parental daphnids was 4 in the control and the tested concentration levels of 0.0432 to 1.83 mg/L.
The first appearance of living juveniles was observed between days 8 and 11 by all parental daphnids of the control and the concentration levels of 0.0432 to 1.83 mg/L. In the concentration level of 6.91 mg/L, all daphnids died before producing juveniles.

• No stillborn juveniles or aborted eggs were observed in the control and the concentration levels of 0.0432 and 0.0693 mg/L during the
exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs
was ≤ 1 % in the concentration levels of 0.138 and 1.83 mg/L, which is negligible.

• The test item induced significant adult mortality of in the concentration levels of 1.83 mg/L (70 %) and 6.91 mg/L (100 %) during the test period
of 21 days. In the control and in the concentration level of 0.0693 mg/L, all parental daphnids survived until the end of the test after 21 days. In the concentration levels of 0.0432 and 0.138 mg/L, an adult mortality of 10 % was observed, which is not biologically significant
(see validity criteria for the control).
The EC50 for the adult mortality was calculated by sigmoidal dose-response regression to be 1.07 mg/L (95 % confidence limits: 0.548 -
1.97 mg/L). The EC100 for the adult mortality was 6.91 mg/L after 21 days.


• The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.0432 to 1.83 mg/L were
determined to be in the range of 4.75 to 4.94 mm per daphnid and 4.98 mm per daphnid in the control group.
The mean dry body weight of the surviving parental daphnids was determined to be in the range of 0.50 to 0.87 mg per daphnid in the
concentration levels of 0.0432 to 1.83 mg/L and 0.88 mg per daphnid in the control.

• No males or ephippia (winter eggs) were observed in the control or in the test groups.

• Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.

• In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to
AQS P 9/2 (02/2000) and OECD 202 is monthly carried out with potassium dichromate as reference item.
The EC50 of the reference item was 2.00 mg/L (Cl: 1.83 - 2.19 mg/L) after 24 hours and is therefore within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the
reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) of the control and the highest tested concentration level of 6.91 mg/L were measured in one replicate. Significant deviations of ≥1.5 units between initial and final pH values were not observed.

The incubator temperature was in the range of 19 to 20 °C.





Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 2.00 (CI 1.83 - 2.19) mg/L
Reported statistics and error estimates:
Statistical evaluation:
The NOEC and the LOEC based on the statistically significant reduction of the reproductive output were deduced from the results of the statistical
evaluation as described below.
Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test
and analysis of variance.
Prior to running the analysis of variance, a normality test and an equal variance test was performed. P-values of the normality and equal variance tests
should be 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
The determination of significant deviations of the number of living juveniles per parental daphnid as well as the intrinsic rates of natural increase was
conducted with One Way Analysis of Variance, DUNNETT’S method (P < 0.05).
The statistical evaluation was carried out with the validated software SigmaPlot for Windows, Version 11.0.
The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
Since the measured concentrations did not remain within ± 20 % of the nominal or initially measured concentrations, all effect values given are based
on the geometric mean measured concentrations of the test item.

Determination of the EC-values:
The EC10- and the EC50-value value for the reduction of the reproductive output and the EC50-value for the adult mortality were calculated by
sigmoidal dose-response regression. The respective 95 % confidence limits for the EC-values were calculated from the standard error and
the t-distribution. All calculation were carried out from the best-fit values with the validated software GraphPad Prism5.
The EC50-value for the reference item and its 95 % confidence limits were calculated accordingly.







 Number of living Juveniles in the Control and Test Groups after 21 Days

Geometric

mean meas.

conc.

of the

test item

 

 

 

 

 

 

 

 

 

 

 

No.

of

parents

prod. juv.

Mean no. of juveniles

per parent

producing juveniles

 

Comparison

versus

control

Number of living juveniles in replicate no.

Total

no.

CV

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Red.

Stat.

[mg/L]

1

2

3

4

5

6

7

8

9

10

å

N

MV±SD

[%]

[%]

 

6.91

--

--

--

--

--

--

--

--

--

--

   0

 0

--

 

--

100

--

1.83

--

--

64

--

60

--

--

--

--

73

197

 3

66±

7

10

21

yes

0.138

 92

73

91

78

82

--

77

73

79

75

720

 9

80±

7

 9

 4

no

0.0693

102

90

89

89

90

88

94

82

91

73

888

10

89±

8

 8

-6

no

0.0432

 81

84

83

77

94

78

94

--

84

83

758

 9

84±

6

 7

-1

no

Control

 85

81

82

91

85

84

79

75

90

83

835

10

84±

5

 6

meas. conc. = measured concentration

N                   = number of the surviving parental daphnids producing living juveniles

Red.              = reduction of the reproductive output

Stat.              = statistical significance(One Way Analysis of Variance,Dunnett’sMethod,P < 0.05)

yes                = statistically significant

no                = statistically not significant

--                   = not applicable, due to the mortality of the parental daphnid(s)

 Intrinsic Rates of Natural Increase

Geometric mean

measured concentration

of the test item

[mg/L]

Intrinsic rate of natural increase in replicate no.

Mean IR

CV

1

2

3

4

5

6

7

8

9

10

MV±SD

[%]

6.91

--

--

--

--

--

--

--

--

--

--

--

 

--

1.83

--

--

0.41

--

0.39

--

--

--

--

0.35

0.38±

0.031)

8

0.138

0.43

0.47

0.47

0.44

0.50

--

0.44

0.47

0.43

0.47

0.46±

0.02

5

0.0693

0.47

0.47

0.46

0.45

0.46

0.45

0.47

0.49

0.47

0.47

0.47±

0.01

2

0.0432

0.43

0.45

0.43

0.41

0.43

0.45

0.46

--

0.37

0.49

0.43±

0.03

8

Control

0.45

0.44

0.44

0.45

0.45

0.43

0.43

0.44

0.45

0.44

0.44±

0.01

2

--   = not applicable, due to the mortality of the parental daphnid(s)

1)   = statistical significance (One Way Analysis of Variance,Dunnett’sMethod, P < 0.05)

 

 First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Geometric mean

measured concentration

of the test item

[mg/L]

Day of first appearance of living juveniles

First

Mean

in replicate no.

appearance

number

1

2

3

4

5

6

7

8

9

10

mean day

of broods

6.91

--

--

--

--

--

--

--

--

--

--

--

--

1.83

--

--

9

--

9

--

--

--

--

11

9.7

4.0

0.138

9

8

9

9

8

--

9

8

 9

 8

8.6

4.0

0.0693

9

9

9

9

9

9

9

8

 8

 8

8.7

4.0

0.0432

9

9

9

9

9

8

9

--

11

 8

9.0

4.0

Control

9

9

9

9

9

9

9

9

 9

 9

9.0

4.0

--   = not applicable, due to the mortality of the parental daphnid(s)

 

Stillborn Juvenilesand Aborted Eggs after 21 Days

Geometric mean

measured concentration

of the test item

[mg/L]

Number of

Total no.

 

Mean stillborn juveniles and aborted eggs per parent alive

MV±SD

stillborn

aborted

juveniles

eggs

å

N

6.91

--

--

--

 0

--

1.83

1

0

1

 3

0.33±0.58

0.138

0

8

8

 9

0.89±1.54

0.0693

0

0

0

10

0±0

0.0432

0

0

0

 9

0±0

Control

0

0

0

10

0±0

N      = number of the surviving parental daphnids producing living juveniles

 

  Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juveniles

Geometric mean

measured concentration

of the test item

[mg/L]

Number of juveniles

Percentage of

dead juveniles#)

 

[%]

Dead

Alive

Total

6.91

--

--

--

--

1.83

1

197

198

< 1

0.138

8

720

728

  1

0.0693

0

888

888

  0

0.0432

0

758

758

  0

Control

0

835

835

  0

Dead= aborted eggs + stillborn juveniles

Total= dead + alive juveniles

#)       = related to the total number of juveniles

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the significant reduction of the reproductive output at the concentration level of 1.83 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 0.138 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 1.83 mg/L. The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 1.02 mg/L (95 % confidence limits: < 0.0432 - 3.11 mg/L).


Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 days) of the test substance was conducted according to OECD 211 (2012) from 2015-08-19 to 2015-09-10, with the definitive exposure phase from 2015-08-19 to 2015-09-09, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

Ten daphnids (less than 24 hours old at the start of the test) were used and individually held in ten replicates per concentration level and control. The study was carried out under semi-static conditions with a renewal of the test solutions 3 times per week (i.e. on Monday, Wednesday and Friday). Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality and body length of the parental daphnids.

Based on the results of a non GLP preliminary range finding test, the study was carried out with 5 concentrations levels in the range of 0.205 to 8.00 mg/L. The test item concentrations were as follows: 0.205-0.512-1.28-3.20-8.00 mg/L. The concentration levels of the test item were analytically verified by SPME-GC-MS at the start of the exposure intervals on days 0, 7, 9, 14, 19 and at the end of the exposure intervals on days 2, 9, 12, 16, 21in all concentration levels and the control. The measured concentrations of the test item at the start of the exposure intervals (0 hours) were in the range of 81 to 106 % of the nominal values. At the end of the exposure intervals (after 48 and 72 hours), the measured concentrations of the test item were in the range of < LOQ to 88 % of the nominal values. The geometric mean measured concentrations of the test item were calculated and used for estimation of the effect levels, because the test item concentrations at the end of the exposure intervals did not remain stable within the recommended range of ±20 % of the nominal or initially measured concentrations. The geometric mean measured concentrations of the test item are as follows: 0.0432 - 0.0693 - 0.138 - 1.83 - 6.91 mg/L.

 

·  The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 84 in the control group. The reproductive output was statistically significant reduced at the concentration level of 1.83 mg/L (21.3 %) in comparison to the reproductive output in the control (One Way Analysis of Variance,Dunnett’sMethod, P < 0.05). In the concentration levels of 0.0432 to 0.138 mg/L, the reproductive output was comparable to the reproductive output in the control. In the concentration level of 6.91 mg/L, all daphnids died before producing juveniles and therefore the reduction of the reproductive output was set to 100 %.

The EC10for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 1.02 mg/L (95 % confidence limits: < 0.0432 - 3.11 mg/L). The EC50for the reduction of the reproductive output was calculated accordingly to be 3.70 mg/L (95 % confidence limits: 2.12 - 5.37 mg/L).


 

·  Based on the significant reduction of the reproductive output at the concentration level of 1.83 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 0.138 mg/L andtheLowest Observed Effect Concentration (LOEC) was assessed at 1.83 mg/L.

 

·  The coefficient of variation of thenumber of living offspring produced per surviving parentaldaphnid was 6 % inthe control. At the concentration levels of 0.0432 to 1.83 mg/L, the coefficients of variation were in the range of 7 to 10 % (Table1andTable3). Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be 25 %.

 

·  The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of natural increase determined for the surviving daphnids at the concentration level of 1.83 mg/L was statistically significant in comparison to the IR at the control (One Way Analysis of Variance,Dunnett’sMethod, P < 0.05). At the concentration levels of 0.0432 to 0.138 mg/L, the IR were comparable to the IR at the control. Due to 100 % adult mortality, no IR could be calculated at the concentration level of 6.91 mg/L. For details, seeTable1,Table4 and Figure 3.

 

·  At the end of the test after 21 days, the number of broods with living juveniles produced by the surviving parental daphnids was 4 in the control and the tested concentration levels of 0.0432 to 1.83 mg/L.
The first appearance ofliving juveniles was observed between days 8 and 11 by all parental daphnids of the control and the concentration levels of 0.0432 to 1.83 mg/L.In the concentration level of 6.91 mg/L, all daphnids died before producing juveniles.

 

·  No stillborn juveniles or aborted eggs were observed in the control and the concentration levels of 0.0432 and 0.0693 mg/L during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was 1 % in the concentration levels of 0.138 and 1.83 mg/L, which is negligible.

 

·  The test item induced significant adult mortality of in the concentration levels of 1.83 mg/L (70 %) and 6.91 mg/L (100 %) during the test period of 21 days.In the control and in the concentration level of 0.0693 mg/L, all parental daphnids survived until the end of the test after 21 days. In the concentration levels of 0.0432 and 0.138 mg/L, an adult mortality of 10 % was observed, which is not biologically significant (see validity criteria for the control).

The EC50for the adult mortality was calculated by sigmoidal dose-response regression to be 1.07 mg/L (95 % confidence limits: 0.548 - 1.97 mg/L). The EC100for the adult mortality was 6.91 mg/L after 21 days.

 


 

·  The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.0432 to 1.83 mg/L were determined to be in the range of 4.75 to 4.94 mm per daphnid and 4.98 mm per daphnid in the control group.

    The mean dry body weight of the surviving parental daphnids was determined to be in the range of 0.50 to 0.87 mg per daphnid in the concentration levels of 0.0432 to 1.83 mg/L and 0.88 mg per daphnid in the control.

·      No males or ephippia (winter eggs) were observed in the control or in the test groups.

 

·  Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.

 

·  In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to AQS P 9/2 (02/2000) and OECD 202 is monthly carried out with potassium dichromate as reference item.
The EC50of the reference item was
2.00 mg/L (Cl: 1.83 - 2.19 mg/L) after 24 hours and is therefore within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according toAQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.


 

A summary of all test item related effects and the assessed effect levels based on the geometric mean measured concentrations of the test

item is given in the table below:

 

   Test Item Related Effects, NOEC, LOEC, EC10, EC50and EC100

(based on the geometric mean measured concentrations of the test item)

Effects

GAMMA UNDECALACTONE

Geometric mean measured concentrations
of the test item
(nominal test item concentrationsare given in italic font)

[mg/L]

 

Control

0.0432

(0.205)

0.693

(0.512)

0.138

(1.28)

1.83

(3.20)

6.91

(8.00)

Mean Number of Living Juveniles per Producing

Parental Daphnid (Reproduction Rate±SD)

84±5

84±6

89±8

80±7

66±71)

---

Coefficient of Variation of theMean Number

of Juveniles per Producing Parent [%]

 6

 7

 8

 9

10

---

Mean Intrinsic Rates of Natural Increase

 0.44

 0.43

 0.47

 0.46

 0.381)

---

First Appearance of Living Juveniles [Mean Day]

 9.0

 9.0

 8.7

 8.6

 9.7

---

Mean Number of Broods with Living Juveniles

 4.0

 4.0

  4.0

  4.0

  4.0

---

Percentage of Dead Juveniles Related to the

Total Number of Juveniles [%]

 0

 0

 0

 1

  1

---

Parental Daphnids: Mean Dry Weight [mg]

 0.88

 0.77

  0.75

 0.87

 0.50

---

Parental Daphnids: Mean Body Length [mm]

 4.98

 4.94

  4.93

  4.83

 4.75

---

Adult Mortality after 21 Days [%]

 0

10

  0

 10

702)

1002)

EC50 Adult mortality(with 95 %confidence limits)

1.07 (Cl: 0.548 - 1.97)

EC100 Adult mortality

6.91

EC10 Reproduction(with 95 %confidence limits)

1.02 (Cl: < 0.0432 - 3.11)

EC50 Reproduction(with 95 %confidence limits)

3.70 (Cl: 2.12 - 5.37)

NOECReproduction

0.138

LOECReproduction

1.83

1)          = statistical significance (One Way Analysis of Variance,Dunnett’sMethod, P < 0.05)

2)          = biologically significant adult mortality observed (> 20 %)

---        = not applicable, due to 100 % adult mortality

Description of key information

OECD Guideline 211, GLP, key study, validity 2:

21d-EC10 (Daphnia magna), reproduction = 1.02 mg/L (95% CL: <0.0432 - 3.11 mg/L) based on geometric mean measured concentrations.

Key value for chemical safety assessment

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

Additional information

One key study is available to asses the long-term toxicity of the registered substance to aquatic invertebrates, according to OECD Guideline 211 with GLP statement. This study is considered reliable with restrictions as one deviation was observed. Indeed, according to the most recent OECD Guideline 211 (2012), the response variable number of living offspring produced per surviving parental animal has been supplemented with an additional response variable for Daphnia reproduction: “total number living offspring produced at the end of the test per parent daphnia at the start of the test (excluding from the analysis parental accidental and/or inadvertent mortality)” but this is not mentioned in the report. This deviation is not considered to have a negative impact on the quality of this study.

Daphnia magna were exposed to five test substance concentrations: 0.205, 0.512, 1.28, 3.2 and 8.0 mg/L, plus one control, during 21 days under semi-static conditions (renewal of the test solutions 3 times per week) in order to assess effects on the reproduction capacity and other test item-related effects or parameters (such as intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality and body length of the parental daphnids).

All test substance concentrations (and the control) were analytically verified by SPME-GC-MS at the start of the exposure intervals on days 0, 7, 9, 14, 19 and at the end of the exposure intervals on days 2, 9, 12, 16, 21. The measured concentrations of the test substance at the start of the exposure intervals (0 hours) were in the range of 81 to 106 % of the nominal values. At the end of the exposure intervals (after 48 and 72 hours), the measured concentrations of the test substance were in the range of < LOQ to 88 % of the nominal values. The geometric mean measured concentrations of the test substance, calculated at 0.0432, 0.0693, 0.138, 1.83 and 6.91 mg/L, were used for estimation of the effect levels, because the test substance concentrations at the end of the exposure intervals did not remain stable within the recommended range of ± 20 % of the nominal or initially measured concentrations.

According to the results of this study, the test substance induced signficant adult mortality of the concentration levels of 1.83 mg/L (70%) and 6.91 mg/L (100%) during the test period of 21 days. In the control and in the concentration level of 0.0693 mg/L, all parental daphnids survived until the end of the test after 21 days. In the concentration levels of 0.0432 and 0.138 mg/L, an adult mortality of 10% was observed, which is not biologically significant. The reproduction output, determined by the average number of living juveniles per surviving parental daphnid at the end of the test after 21 days, was statistically significant reduced at the concentration level of 1.83 mg/L (21.3%) in comparison to the reproductive output in the control. In the concentration levels of 0.0432 to 0.138 mg/L, the reproduction output was comparable to the reproduction output in the control. In the concentration level of 6.91 mg/L, all daphnids died before producing juveniles and therefore the reduction of the reproductive output was set to 100%.

In conclusion, based on the significant reduction of the reproduction output at the concentration level of 1.83 mg/L, the 21d-NOEC and 21d-LOEC were assessed at 0.138 mg/L and 1.83 mg/L, respectively. The 21d-EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 1.02 mg/L (95% CL: <0.0432 - 3.11 mg/L).

NOECs are increasingly contested in ecotoxicology. Indeed, the NOEC is determined as the concentration directly below the LOEC and depend upon the choice made by the Study Director and not a statistically interpolated value. So, NOEC may occur at concentrations considerably lower than the point at which no biological effect occurs. The choice of the concentrations tested is therefore a limiting factor for the NOEC determination. To use a less selective method, ECx approach was taken into account. EC10 values are accepted for risk assessment purposes and CLP allows the use of both NOEC/ECx (e.g. EC10) for classification to determine long-term effects, and are considered as the best endpoint for long-term data. For this reason, the 21d-EC10 value at 1.02 mg/L was considered as the key value for the long-term toxicity of the registered substance to aquatic invertebrates.