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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2010-01-13 to 2010-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study. For justification of read-across within the category please see chapter 1 of the Chemical Safety Report.

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Licowax S
- Physical state: Solid, pale yellow powder
- Storage condition of test material: Refrigerator, 6 +/- 2 °C, protected from moisture and light

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: 100 mg test item/L was weighed out. The WAF was prepared with dilution water and shaken with 20 rpm for 48 h at room temperature. After a separation phase of at least 1 h the WAF was taken from the homogeneous liquid phase.
- Eluate: Dilution water
- Differential loading: Water accommodated fraction (WAF) with a nominal loading of 100 mg/L 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:
Nominal Loading of the WAF
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
100 167 168 180 187 180 173
Control 180 170 164 189 180 175
Test temperature:
Nominal Loading of the WAF
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
100 21.1 22.0 20.1 20.5 20.0 21.0
Control 20.0 21.5 19.1 20.6 19.3 21.0
pH:
Nominal Loading of the WAF
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
100 7.53 7.45 7.42 7.46 7.52 7.45
Control 7.92 7.47 7.93 7.14 7.87 7.18
Dissolved oxygen:
Nominal Loading of the WAF
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Jan. 13th Jan. 15th Jan. 20th Jan. 22nd Jan. 27th Jan. 29th
100 8.03 7.01 7.68 7.41 8.94 9.93
Control 8.36 6.57 8.49 7.29 9.17 6.07
Salinity:
Not measured, freshwater
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x 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.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1

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

RANGE-FINDING STUDY
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal Loading
of the
Water Accommodated Fraction (WAF)
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
100 0 0 0 0 0 0
10 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:
NOELR
Effect conc.:
100 other: mg/L (loading of the water accomodated fraction)
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 WAF
- 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 111 juveniles at the WAF 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: None
- Type and number of behavioural abnormalities: None
- 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 between day 7 and day 8
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.20 (CI 1.08 - 1.34)
Reported statistics and error estimates:
The NOEL and LOEL for reproduction as measured by the intrinsic rate of natural increase and total living neonates per surviving female as well as adult survival were determined directly from the results. Significant deviations were determined in comparison to control using statistical standard procedures as normality test, equal variance test and t-test.
Prior to running a t-test a normality test and an equal variance test were performed. P-values for both normality and equal variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
For the determination of significant deviations for the reproduction rates, the intrinsic rates of natural increase and the total body lengths t-test (p = 0.05) was used.
The coefficients of variation around the mean number of living offspring produced per parent at the control group and the saturated solution were evaluated.

Any other information on results incl. tables

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

Nominal Loading of the WAF

[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

Red.

Sum

N

MV ± SD

[%]

[%]

100

105

102

134

  87

109

127

101

122

98

121

1106

10

111 ±

15

13

4

Control

153

  95

  97

123

140

124

124

103

97

104

1160

10

116 ±

20

17

-

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

                        (n = 10)

Nominal Loading
of the WAF

[mg/L]

Mortality [%]

7 days

14 days

21 days

100

0

0

0

Control

0

0

0

First Appearance of Living Juveniles in the Individual Groups

Nominal Loading
of the WAF

[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

100

7

8

8

8

8

8

7

8

7

8

7.7

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 WAF

[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

100

5.25

5.00

5.25

5.00

5.00

5.50

5.25

5.75

4.50

5.00

5.15

10

7.6

0.76

Control

5.50

5.50

5.00

5.50

5.50

5.00

5.25

5.00

4.75

5.25

5.23

10

7.7

0.77

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
At the water accommodated fraction ((WAF) of the test item Licowax S with a nominal loading of 100 mg/L no biologically or statistically significant effects were determined. The No Observed Effect Loading (NOEL) and the Lowest Observed Effect Loading (LOEL) were assessed at:
NOEL: Water Accomodated Fraction (with the nominal loading of 100 mg/L)
LOEL: > Water Accomodated Fraction (with the nominal loading of 100 mg/L)
All effect values given are based on the WAF of Licowax S with the nominal loading of 100 mg/L.
Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of Licowax S was conducted as a limit test with the water accommodated fraction (WAF)* according to OECD 211 (2008) from 2010-01-13 to 2010-02-04, with the definitive exposure phase between 2010-01-13 to 2010-02-03, 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 h 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
Licowax S was not carried out, due to the low solubility of the test item in water. All results given are based on the water accommodated fraction (WAF)* with the nominal loading of 100 mg/L.

·   The average number of juveniles per parental daphnid in the control group was 116 and 111 at the WAF* after 21 days. The reproductive output at the limit concentration is comparable to the control group (t-test, p = 0.05). Details are presented in Table 1, Table 3 and Figure 1.

·   The coefficient of variation of the number of living offspring produced per parent was 17 % at the control group and 13 % at the WAF*.

·   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 WAF* was comparable to the control group (t-test, p = 0.05).

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

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the WAF*, 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 8 by all parent animals of the control and the WAF.

·   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 0.77 mg per daphnid at the control group and 0.76 mg per daphnid at the WAF. The mean body length of the parental daphnids at the WAF was 5.15 mm per daphnid and 5.23 mm per daphnid at the control group.

·   No malesand ephippia (winter eggs) were observed at the 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 WAF with the nominal loading of 100 mg/L of the test item Licowax S.

Table 1:    Summary of all Test Item Related Effects

                   (Based on the WAF of Licowax S with the nominal loading of 100 mg/L)

Effects

Licowax S

Significance

Control

Water Accommodated Fraction (WAF)

(Nominal Loading 100 mg/L)

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

116 ± 20

111 ± 15

no

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

  17

  13

no

Intrinsic Rate of Natural Increase

    0.48

    0.47

no

Appearance of First Brood [Mean Day]

    8.0

    7.7

no

Number of Broods

    5

    5

no

Stillborn Juveniles and Aborted Eggs

    0

    3

no

Adult Mortality after 21 days [%]

    0

    0

no

Parent Animals: Mean Dry Weight [mg]

    0.77

    0.76

no

Parent Animals: Mean Body Length [mm]

    5.23

    5.15

no

NOELReproduction

Water Accommodated Fraction (WAF)

(Nominal Loading 100 mg/L)

LOELReproduction

> Water Accommodated Fraction (WAF)

(Nominal Loading 100 mg/L)

SD    = Standard deviation

no     = No biological significance

 


 

·   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 1.20 mg/L after 24 h 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.