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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-09 to 2009-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (10 mg/L test item) was prepared with dilution water two days prior to application. The stock solution was shaken with 20 rpm for 48 h. Undissolved particles were removed by centrifugation with 10000 rpm for 10 min.
- Eluate: Dilution water
- Differential loading: Saturated solution as limit concentration
- Controls: 10 replicates of dilution water without test item.

Test organisms

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
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
Not observed

Test conditions

Hardness:
Total Hardness as CaCO3 [mg/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 170 182 177 178 175 174
Control 175 185 177 198 180 173
Test temperature:
Temperature [°C]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 22.0 20.8 21.9 20.7 21.9 20.6
Control 21.2 21.1 21.8 21.0 21.8 20.6
pH:
pH-Values
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 7.54 7.73 7.70 7.54 7.74 7.64
Control 7.87 7.77 7.97 7.85 7.94 7.66
Dissolved oxygen:
Dissolved Oxygen Concentration [mg O2/L]
I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Nov. 11th Nov. 13th Nov. 18th Nov. 20th Nov. 25th Nov. 27th
Saturated
Solution 8.05 8.78 8.62 8.56 7.62 6.90
Control 8.10 9.24 8.04 8.11 8.10 6.48
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
Please refer to information in materials and methods incl. tables
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): slightly closed with small glass plates
- Material, size, headspace, fill volume: Glass beakers (5 x 8 cm), 100 mL capacity, 50 mL fill volume
- Aeration: No additional aeration
- Renewal rate of test solution (frequency/flow rate): semi-static, medium renewal 3 times per week
- 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
- Total organic carbon: not determined
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: Not necessary
- Photoperiod: 16:8 h light:dark cycle
- Light intensity: max. 20 µExm-2xs-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Dissolved oxygen concentration, pH-values, total hardness and water temperature were measured once every 7 days, in fresh and old media, in one replicate of the control and the limit concentration.
Mortality of parental daphnids
The number of parental daphnids which were immobilized or dying during the test and day of death was observed and recorded once a day at least at the same time as the offspring is counted. Dead specimens were removed.
Neonates
First appearance of juveniles was checked daily.
The number of neonates (alive and dead progeny) was counted and dates of release were recorded once a day from the first day of production of neonates until the end of the test. The neonates (alive and dead) were removed after counting to prevent from consuming food intended for the adults. The number of aborted eggs and dates of observation was recorded.
Condition of parental daphnids
Abnormalities (e.g. swimming behaviour, number of males and winter eggs) were observed and recorded at each day of observation.
Total length and dry body weight of parental daphnia
At the end of the test total length of each daphnid and the mean dry body weight of the daphnids alive at the saturated solution and control were determined. The mean dry body weight of a surviving daphnid was calculated from the dry mass of all daphnids alive at the saturated solution and control.

VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: 1:1, 1:10, 1:100
- Results used to determine the conditions for the definitive study:
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Dilution level
of the
saturated solution IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
1:1# 0 0 0 0 0 0
1:10 10 0 5 10 0 5
1:100 0 0 0 0 0 0
Control 0 0 0 0 0 0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
> 10 other: mg/L (nominal loading) saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution
- No. of offspring produced per day per female: The mean number of offspring alive produced per parent animal surviving at the end of the test was 116 juveniles at the control group and 117 juveniles at the saturated solution after 21 days. The reproductive output at the limit concentration is comparable to the control group
- Body length and weight of parent animals: please refer to the respective table
- Type and number of morphological abnormalities: no morphological abnormalities detected
- Type and number of behavioural abnormalities: no behavioural abnormalities detected
- Number of males and females (parental): No males were observed in either the control or the test group during the test.
- Time to first brood release or time to hatch: The first day of appearance of juveniles at the test and control group was day 8
- Other biological observations: No ephippia were observed in the control or in the test group during the test. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 %, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.67 (CI 1.63 - 1.71)
Reported statistics and error estimates:
NOEC (No observed The highest tested concentration of the test item at which the test
effect concentration) item is observed to have no statistically or biologically significant effect when compared to the control.

LOEC (Lowest observed The lowest tested concentration of the test item at which the test
effect concentration) item is observed to have a statistically or biologically significant effect when compared to the control.

Any other information on results incl. tables

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                        (n = 10)

Nominal Loading
of the
Saturated Solution
[mg/L]

Mortality [%]

7 days

14 days

21 days

10.0

0

0

0

Control

0

0

0

Number of Juveniles in the Control and Test Group after 21 Days

Nominal Loading of the Saturated
Solution

[mg/L]

No.

Mean No. of Juveniles

Compared

Number of Juveniles in Replicate No.

Total

of

CV

versus

No.

Parents

per Parent

Control

prod. Juv.

producing Juveniles

Inc.

Sum

N

MV ± SD

[%]

[%]

10.0

117

107

119

121

128

112

108

114

123

116

1165

10

117 ±

 7

6

1

Control

102

124

116

126

115

108

118

121

118

114

1162

10

116 ±

 7

6

--

N= number of parents producing juveniles alive; Inc.= increase of reproductive output

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading
of the
Saturated Solution
[mg/L]

Day of First Appearance of Living Juveniles

First

in Replicate No.

Appearance

1

2

3

4

5

6

7

8

9

10

Mean Day

10.0

9

8

8

8

9

8

9

9

8

8

8.4

Control

8

8

8

8

8

8

8

8

8

8

8.0

Total Length and Dry Weight of the Parental Daphnids

Nominal Loading
of the
Saturated Solution
[mg/L]

Total Length of the Parental Daphnids [mm]

MV

N

Dry Weight [mg]

Replicate No.

1

2

3

4

5

6

7

8

9

10

[mm]

S

MV

10.0

5.00

5.50

5.25

5.00

5.75

5.50

5.25

5.50

5.50

5.25

5.35

10

8.6

0.86

Control

5.50

5.50

5.00

5.75

5.50

5.00

5.75

5.25

5.00

5.50

5.38

10

8.7

0.87

S= sum; N= number of parental daphnids alive;

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
At the saturated solution of the test item with a nominal loading of 10.0 mg/L no biologically or statistically significant effects were determined. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were assessed at:
NOEC: Saturated Solution (with the nominal loading of 10.0 mg/L)
LOEC: > Saturated Solution (with the nominal loading of 10.0 mg/L)
All effect values given are based on the saturated solution of the test item with the nominal loading of 10 mg/L.
Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) with the test item was conducted as a limit test with the saturated solution* according to OECD 211 (2008) from 2009-11-09 to 2009-12-03 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, Germany.
Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held were used for the limit concentration and control. At test start they were 2 to 24 hours old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as occurrence of aborted eggs and stillborn juveniles, time of production of first brood, adult mortality, intrinsic rate of natural increase, dry body weight and length of the parent animals.
A specific analytic of the test item
was not carried out, due to the low solubility of the test item in water. All results given are based on the saturated solution* with the nominal loading of 10 mg/L.

·   The average number of juveniles per parental daphnid in the control group was 116 and 117 at the saturated solution* after 21 days. The reproductive output at the limit concentration is comparable to the control group (Unpaired t-test, p = 0.05).

·   The coefficient of variation of the number of living offspring produced per parent was 6 % at the control group and the saturated solution*.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The IR of the saturated solution* was comparable to the control group (Unpaired t-test, p = 0.05).

·   No adult mortality or immobilization of parental daphnids was observed at the control group and at the saturated solution*.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the saturated solution, which is seen to be not biologically significant. At the control no dead juveniles or aborted eggs were observed.

Five broods were observed during the test period at all surviving parental daphnids. The first brood was released till day 9 by all parent animals of the control and the saturated solution*.

·   The mean dry body weight and total body lengths of all parental daphnids of the test and control group determined at the end of the study were comparable.
The mean dry body weight of the parental daphnids was 0.87 mg per daphnid at the control group and 0.86 mg per daphnid at the saturated solution. The mean body length of the parental daphnids at the saturated solution was 5.35 mm per daphnid and 5.38 mm per daphnid at the control group.

·   No males and ephippia (winter eggs) were observed at control or test groups.

A summary of all test item related effects is given in Table 1. There was no biologically or statistically significant effect determined at the saturated solution* of the test item.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the saturated solution* of test item with the nominal loading of 10 mg/L)

Effects

Test item

Significance

Control

Saturated Solution*

(Nominal Loading 10 mg/L)

Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)

116 ± 7

117 ± 7

no

Coefficient of Variation of the Mean Number of Juveniles per Producing Parent

    6

    6

no

Intrinsic Rate of Natural Increase

    0.49

    0.47

no

Appearance of First Brood [Mean Day]

    8.0

    8.4

no

Number of Broods

    5

    5

no

Stillborn Juveniles and Aborted Eggs

    0

  10

no

Adult Mortality after 21 days [%]

    0

    0

no

Parent Animals: Mean Dry Weight [mg]

    0.87

    0.86

no

Parent Animals: Mean Body Length [mm]

    5.38

    5.35

no

NOECReproduction

Saturated Solution*

(Nominal Loading 10 mg/L)

LOECReproduction

> Saturated Solution*

(Nominal Loading 10 mg/L)

The water quality parameters (pH-value, dissolved oxygen, water hardness and temperature) were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC50 of the reference item at 1.67 mg/L after 24 hours was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to
AQS P 9/2 (05/1996) 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.
*) Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium , acc. to OECD Series No. 23 2000(6)