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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:
supporting study
Study period:
2009-04-24 to 2010-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, Identity data complete, incuding chemical analyses.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
(Z)-N-(3-aminopropyl)-N'-9-octadecenylpropane-1,3-diamine
EC Number:
249-276-6
EC Name:
(Z)-N-(3-aminopropyl)-N'-9-octadecenylpropane-1,3-diamine
Cas Number:
28872-01-7
IUPAC Name:
N-(3-aminopropyl)-N'-octadec-9-en-1-ylpropane-1,3-diamine
Details on test material:
- Chemical name: N-(3-aminopropyl)-N'-octadec-9-en-1-ylpropane-1,3-diamine
- EC number: 249-276-6

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Two concentration levels 30 and 270 µg/L and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 h). For the longest exposure interval of 72 h samples were taken from the fresh (0 h) and old media (72 h) once within the test period, respectively. For the first sampling interval samples of fresh (0 h) and old media (48 h) of the concentration levels 30.0, 90.0 and 270 µg/L were taken and analysed. For the analyses of the old media separate replicates without algae and test organisms were prepared and stored under test conditions. Additionally, the freshly prepared stock solution of 10 mg/L was analytically verified at least once within 7 days to check their correct preparation. The adsorption of the test item was quantified exemplarily. Therefore, two test replicates containing daphnids and algae of the test concentration 270 mg/L, containing daphnia and algae, were prepared analogously to the test design of the definitive test, emptied after 72 h of exposure and rinsed twice with demineralised water. Thereafter an extraction of the test vessel was carried out. The test item concentration in the fresh (0 h) and old media (72 h) were analytically verified, too. The analytical method was validated.
- Sample storage conditions before analysis: All samples were stored at room temperature, if necessary

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (10 mg/L) was freshly prepared with natural river water for each water renewal. The stock soltion was treated with ultrasound for 15 min at 40 °C.
- Eluate: Natural river water
- Differential loading: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L
- Controls: 10 replicates of natural 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:
no
Total exposure duration:
21 d
Post exposure observation period:
Not observed

Test conditions

Hardness:
Total Hardness [mg/L] as CaCO3
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
May 20th May 22nd May 27th May 29th June 3rd June 5th
810 308 271 319 297 342 306
Control 320 292 319 301 317 304
Test temperature:
Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
May 20th May 22nd May 27th May 29th June 3rd June 5th
810 22.0 22.0 21.8 21.7 21.1 21.2
Control 20.3 21.8 19.7 21.5 20.6 21.3
pH:
pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
May 20th May 22nd May 27th May 29th June 3rd June 5th
810 8.41 8.57 7.93 7.68 7.78 7.79
Control 8.24 8.16 7.96 7.67 7.93 7.96
Dissolved oxygen:
Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
May 20th May 22nd May 27th May 29th June 3rd June 5th
810 7.98 10.04 8.74 9.10 9.80 6.56
Control 9.01 7.99 10.19 7.46 9.82 7.13
Salinity:
Not measured, freshwater
Nominal and measured concentrations:
please refer to information on materials and methods
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity
- 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: Natural river water of the river Leine was used. This river is located near D-31171 Nordstemmen, Germany. Specifications of the natural river water are given in the table below.
River: Leine
Location D-31171 Nordstemmen
Sampling Date 2009-05-18 2009-12-15
Dates of use
(experimental phase) 2009-07-22 to 2009-08-12 2010-01-15 to 2010-01-18
(Definitive exposure phase) (Determination of Adsorption)

Weather conditions
on Day of Sampling Dry, few clouds, ca. 19 °C Cloudy, ca -1 °C
Colour Yellowish, slightly turbid Yellowish, clear
pH 7.94 7.97
Conductivity [µS/cm] 736 386
Dissolved Oxygen [mg O2/L 9.02 8.62
DOC [mg C/L] 3.0 3.9
TOC [mg C/L] 3.1 3.9
Ammonium-N [mg N/L] 0.109 0.042
Nitrate-N [mg N/L] 3.43 2.62
Total Nitrogen [mg N/L] 5.23 3.53
o-Phosphate-p [mg P/L] 0.118 0.062
Total Phosphate [mg P/L] 0.141 0.053
Suspended Matter [mg/L] 11.7 16.2
Total Hardness [mg CO3/L] 322 154
Storage conditions 6 ± 2 °C < 10 °C
(Frozen due to low outdoor temperature)





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
- Test concentrations: 10 - 1- 0.1 - 0.01 mg/L

Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 100 100 100 100 100 100
1 100 100 100 100 100 100
0.1 0 0 0 0 10 5
0.01 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 concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
810 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
Adverse effect
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
577 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
684 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult
Remarks on result:
other: (607-770)
Details on results:
- Mortality of parent animals:
The test item induced significant adult mortality of 90 % at the tested concentration level of 810 g/L after 21 days. At the other concentration levels and in the control no significant mortality (The EC50 for adult mortality after 21 days was calculated by sigmoidal dose-response regression to be 684 µg/L (CI: 607 - 770 µg/L). The EC10 for adult mortality after 21 days was calculated to be 577 µg/L.
- 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 136 juveniles in the control group. In the test concentrations 10.0 to 90.0 µg/L the mean number of offspring alive was comparable to the control. The reproductive output increased statistically significant in the concentration levels 270 and 810 µg/L when compared to the control (One Way Analysis of Variance, Dunnett’s method, p = 0.05. For the mean number of living offspring produced per parent in the control group the coefficient of variation amounts to 6 %. At the concentration levels of 10.0 to 270 µg/L the coefficient of variation ranged from 4 to 9 %. For the concentration level of 810 µg/L the coefficient of variation could not be calculated, because only one parent animal survived till the end of the study.
The mean number of juveniles per surviving daphnid of the treatment groups were compared to the control by One Way Analysis of Variance, DUNNETT’S method, p = 0.05. The increase of the reproductive output at the test concentrations 270 µg/L (21 %) and 810 µg/L (22 %) in comparison to the control was statistically significant. The reproductive output at the other concentration levels of 10.0 to 90.0 µg/L was comparable to the control. The EC50 for the reproductive output was determined directly from the results to be > 810 µg/L.
- Body length and weight of parent animals:
At the end of the test the total body length and the dry weight of all surviving parent animals at each tested concentration level and control were determined and given in the respective table.
- Number of males and females (parental):No males were observed in either the control or the test groups during the test.
- Time to first brood release or time to hatch
The first day of appearance of juveniles at all test item and control groups producing juveniles was between day 7 and day 9
- Other biological observations:
Related to the total number of produced juveniles (dead + alive) the fraction of dead juveniles came to a maximum of 3 % in the concentration level 90.0 µg/L. In the control only 3 stillborn juveniles and no aborted eggs were observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.66 (CI 1.50 - 1.83)
Reported statistics and error estimates:
The NOEC and LOEC for the reproduction and the adult mortality as the most sensitive effects were determined directly from the observation data. Significant deviations were determined in comparison to the solvent control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
For the determination of significant deviations for the reproduction rates, the intrinsic rates of natural increase and the body length One Way Analysis of Variance, DUNNETT’S method, p = 0.05 was used. Statistical evaluation of age of first reproduction and occurrence of stillborn juveniles and aborted eggs could not be carried out because Normality Test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control, the solvent control and the test groups were evaluated.
EC50- values of the adult mortality and the reference item were calculated by sigmoidal dose-response regression. The confidence interval for the EC50 of the reference item was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. For the confidence interval of the EC50-value of the adult mortality the highest concentration causing no effect and the lowest concentration with 100 % adult mortality were used as confidence limits, because only one partial effect was observed. The EC50- value of the reproductive output was determined directly from the observation data because reduction of the reproductive output ≥ 50 % was not observed within the tested concentration range.

Any other information on results incl. tables

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

Nominal
conc.

[µg/L]

No.

Mean No. of Juveniles

Comparison

Number of Juveniles in Replicate No.

Total

of

CV

versus

No.

Parents

per Parent

Control

prod. Juv.

producing Juveniles

Inc.

Stat.

  1

  2

  3

  4

  5

  6

  7

  8

  9

   10

å

N

MV ± SD

[%]

[%]

810

--

--

166

--

--

--

--

--

--

--

  166

  1

166 ±

n.a.

n.a.

22

yes

270

145

170

182

184

149

152

168

169

160

162

1641

10

164 ±

13

8

21

yes

    90.0

144

134

132

142

146

149

143

150

140

148

1428

10

143 ±

  6

4

  5

no

    30.0

128

133

149

--

140

123

132

137

109

142

1193

  9

133 ±

12

9

  -2*

no

    10.0

139

145

132

129

140

135

137

121

137

126

1341

10

134 ±

  7

5

  -1*

no

Control

141

139

128

127

142

137

142

122

137

146

1361

10

136 ±

  8

6

First Appearance of Living Juveniles in the Individual Groups

Nominal
concentration

Day of First Appearance of Living Juveniles

First

in Replicate No.

Appearance

[µg/L]

1

2

3

4

5

6

7

8

9

10

Mean Day

810

--

--

8

--

--

--

--

--

--

--

8.0

270

8

8

8

8

8

7

8

8

8

8

7.9

    90.0

8

8

8

8

8

8

8

8

8

8

8.0

    30.0

8

8

8

--

8

8

8

8

9

8

8.1

    10.0

8

8

8

8

8

8

8

8

8

8

8.0

Control

8

8

8

7

8

8

8

7

8

8

7.8

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

                 (n = 10)

Nominal concentration

Adult Mortality [%]

[µg/L]

7 days

14 days

21 days

810

90*

90*

 90*

270

 0

 0

  0

    90.0

 0

 0

  0

    30.0

 0

 0

10

    10.0

 0

 0

  0

Control

 0

 0

  0

Total Body Length and Dry Weight of the Parent Animals

Nominal
concentration

[µg/L]

Total Length of the Parent Animals

[mm]

MV

N

Dry Weight
[mg]

Replicate No.

1

2

3

4

5

6

7

8

9

10

[mm]

S

MV

810

--

--

5.50

--

--

--

--

--

--

--

5.50

  1

    0.60

0.60

270

5.75

6.00

6.00

6.25

5.75

6.00

6.00

5.75

6.00

6.00

5.95

10

12.4

1.24

    90.0

5.75

5.75

5.25

5.75

5.50

5.75

5.75

5.25

5.75

5.75

5.63

10

10.2

1.02

    30.0

5.75

5.50

5.25

--

6.00

6.00

5.50

5.75

5.00

5.75

5.61

  9

    9.40

1.04

    10.0

6.00

5.50

5.75

6.00

5.75

5.75

5.75

6.00

6.00

6.00

5.85

10

11.8

1.18

Control

6.00

6.00

5.75

5.75

5.50

6.00

5.75

5.75

5.25

6.00

5.78

10

11.3

1.13

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study no reduction of the reproductive output, but hormesis was observed. The EC10-value for the increase of the reproductive output, the most sensitive effect in this study, was calculated by sigmoidal dose-response regression. A significant reduction of the reproductive output did not occur. The NOEC was assessed as adverse effect level directly from the observation data taking the observed hormesis not into account. All effect values are given based on the nominal concentrations.

Effect values EC10, Reproduction : 171 µg/L (reproduction increase)
EC50, Reproduction : Not determinable

EC10, Adult Mortality : 577 µg/L
EC50, Adult Mortality : 684 µg/L (Cl: 607 - 770 µg/L)

Adverse effect value NOECReproduction : 810 µg/L

Similar recoveries were obtained for the highest analysed concentration (nominal 270 µg test item/L) of analytical interest. Recoveries between 84 to 87% were obtained in the 2nd to 4th analytical interval for the new media of every batch. A recovery rate of 52% was determined in the first interval. Recoveries between 148 to 167% in fresh media were obtained for the 2nd to 4th analytical interval in the group exposed to 30 µg test item/L (nominal). Analogous to the results determined for the first interval in the test group exposed to 270 µg test item/L only the recovery of the first interval of the nominal test concentration of 30 µg/L with 29% was low. In general lower recoveries were observed for the old media samples independent of the exposure concentration.
Biodegradation as possible reason for the observed decrease of the test concentration during the exposure period is very unlikely considering the short time frame between the refreshments of the test solutions. The concentration of the test item adsorbed to the glassware as determined exemplarily at the test concentration level of 270 µg/L was 40.4 µg/L which corresponds to 15 % of the nominal concentration. The observed concentration decrease between fresh and old media is thus only for a small fraction caused by sorption to the glassware. The main reduction is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Because of the limited sorption to glassware and unlikeliness of biodegradation as possible reason for the reduction of the observed concentration it must be concluded that the test organisms were fully exposed to the bulk concentration of the test substance during the test. All effect values given are therefore based on the nominal test item concentrations.
Executive summary:

The Daphnia magna Reproduction Test with Natural River Water (Semi-Static, 21 d) of the test item Oleyl dipropylene triamine(CAS no. 28872-01-7, batch No. S001256) was conducted according to OECD 211 (2008) from 2009-04-24 to 2010-01-18 with the definitive exposure phase between 2009-05-20 to 2009-06-10 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, .

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At the beginning of the test 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 or parameters such as adult mortality, intrinsic rate of natural increase, time of production of first brood, occurrence of aborted eggs and stillborn juveniles, dry body weight and length of the parent animals.

Oleyl dipropylene triamine (CAS No. 28872-01-7) is poorly soluble in water and also has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain alkyl polyamines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for cationic surface-active substances like the polyamines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with these substances.

The aquatic ecotoxicity tests with polyamines were therefore performed in river water to allow a PECaquatic,bulk/PNECaquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing ‘total aquatic concentrations’. To characterize the risk to the aquatic compartment the PECaquatic,bulk is compared with the PNECaquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2001).

In order to class standard laboratory toxicity study valid, it is of particular importance that - besides information on test substance, test method / conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

For ecotoxicity tests performed using the bulkapproach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier to interpret, more environmental realistic, and if compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

Nominal concentrations of the test item Oleyl dipropylene triamine were selected after a preliminary acute immobilization test (48 h, static) as follows: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L. The test item Oleyl dipropylene triamine was analytically verified in the concentration levels 30.0 and 270 µg/L and in the control via LC-MS/MS of samples taken on days 0, 7, 14, 16 (fresh media) and on days 2, 9, 16 (old media, 48 h) and 19 (old media, 72 h). Additionally the test concentration 90 µg/L was analytically verified at the first sampling interval on day 0 (fresh media) and day 2 (old media, 48 h). The freshly prepared stock solutions of 10 mg test item/L were analytically verified too and gave recoveries in the range of 77 to 89 % of the nominal value. Similar recoveries were obtained for the highest analysed concentration (nominal 270 µg test item/L) of analytical interest. Recoveries between 84 to 87% were obtained in the 2nd to 4thanalytical interval for the new media of every batch. A recovery rate of 52% was determined in the first interval. Recoveries between 148 to 167% in fresh media were obtained for the 2nd to 4th analytical interval in the group exposed to 30 µg test item/L (nominal). Analogous to the results determined for the first interval in the test group exposed to 270 µg test item/L only the recovery of the first interval of the nominal test concentration of 30 µg/L with 29% was low. In general lower recoveries were observed for the old media samples independent of the exposure concentration.

Biodegradation as possible reason for the observed decrease of the test concentration during the exposure period is very unlikely considering the short time frame between the refreshments of the test solutions. The concentration of the test item adsorbed to the glassware as determined exemplarily at the test concentration level of 270 µg/L was 40.4 µg/L which corresponds to 15 % of the nominal concentration. The observed concentration decrease between fresh and old media is thus only for a small fraction caused by sorption to the glassware. The main reduction is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Because of the limited sorption to glassware and unlikeliness of biodegradation as possible reason for the reduction of the observed concentration it must be concluded that the test organisms were fully exposed to the bulk concentration of the test substance during the test. All effect values given are therefore based on the nominal test item concentrations.

·   The average number of juveniles per parent in the control group was 136 after 21 days. The reproductive output increased statistically significant in the concentration levels 270 and 810 µg/L. The reproductive output at the other concentration levels was not statistically significant increased or reduced when compared to the control (One Way Analysis of Variance, Dunnett’s method, p = 0.05). In this study no reduction of the reproductive output, but hormesis was observed. The EC10for the increase of the reproductive output was calculated by sigmoidal dose-response regression to be 171 µg/L. An EC50-value for the reproductive output could not be determined because no effects ≥ 50 % (reduction or increase of the reproductive output) were observed within the tested concentration range.  As adverse effect the NOEC was assessed to be 810 µg/L (hormesis not taken into account).

·   The coefficient of variation of the mean number of living offspring produced per parent in the control group was 6 %.

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 surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance, Dunnett’s method(p = 0.05). There was a statistical significance in the concentration levels 30.0 and 810 µg/L when compared to the control. The statistical significance at the concentration level 30.0 µg/L is seen to be not biologically relevant, because the statistical effect is not concentration related. There was no statistical significance determined at the concentration levels above (90 and 270 µg/L) and below (10 µg/L).

·   Only 3 stillborn juveniles but no aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles came to a maximum of 3 % in the concentration level 90.0 µg/L.

·   The mean day of release of the first brood was day 7.8 in the control group. In the tested concentration levels 10.0 to 810 µg/L the mean day of release of the first brood was in the range of 7.9 to 8.1. The first brood was released until day 9 by all surviving daphnids of the control group and the tested concentration levels. Five broods were released by all surviving animals of the test and control groups.

The statistically significant increase of the reproductive output at the concentration levels 270 and 810 µg/L was the most sensitive effect in this study. A summary of all test item related effects on reproduction is presented in Table 1. The NOEC was assessed as adverse effect level directly from the observation data taking the observed hormesis not into account.

Table 1:        Test Item Related Effects on Reproduction, NOEC and EC50

Effects

Nominal Concentration [µg/L]

Control

10.0

30.0

90.0

270

810

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

136 ± 8

134 ± 7

133 ± 12

143 ± 6

164 ± 131)

166 ± 01)

Mean Intrinsic Rates of Natural Increase

0.52

0.51

0.492)

0.51

0.51

0.461)

Appearance of First Brood [Mean Day]

7.8

8.0

8.1

8.0

7.9

8.0

Number of Broods

5

5

5

5

5

5

EC50, Reproduction

with Confidence Interval (CI) p = 95 %

Not determinable

(CI: Not applicable)

NOEC (adverse effect)

810 µg/L3)

A summary of test item related effects concerning the adult mortality and growth (weight, length) is presented in Table 2.

·   The test item induced significant adult mortality of 90 % in the tested concentration level 810 µg/L after 21 days. At the other concentration levels and in the control no significant mortality (£ 20 %) of parent animals was observed. In the control as well as at the test concentrations 32.0 and 320 µg/L all daphnids survived until the end of the study. The EC10- and the EC50-value for adult mortality after 21 days was calculated applying sigmoidal dose-response regression. The EC50-value was calculated to be 684 µg/L (CI: 607 - 770 µg/L). The EC10-value was calculated to be 577 µg/L.

·   The mean dry body weight and mean total body length of the parent daphnids in the tested concentration levels were comparable to the control group.
The mean dry body weight at the concentration levels 10.0 to 270 µg/L ranged from 1.02 to 1.24 mg per daphnid. The mean dry body weight of only one surviving daphnid at the concentration level of 810 µg/L was determined to be 0.60 mg. Due to the low weight of only one daphnid a comparison with the control group is not useful for the concentration level 810 µg/L. The mean dry body weight in the control was 1.13 mg per daphnid.
The mean total body length in the tested concentrations was in the range of 5.50 to 5.95 mm per daphnid and 5.78 mm per daphnid in the control group.

Table 2:    Test Item Related Effects on Adult Mortality (EC10 and EC50) and Growth (Weight, Length)

Effects

Nominal Concentration [µg/L]

Control

10.0

30.0

90.0

270

810

Adult Mortality after 21 Days [%]

0

0

10

0

0

901)

Parent Animals: Mean Dry Weight [mg]

1.13

1.18

1.04

1.02

1.24

0.60

Parent Animals: Mean Body Length [mm]

5.78

5.85

5.61

5.63

5.95

5.50

EC10, Adult Mortality

577 µg/L

EC50, Adult Mortality

with Confidence Interval (CI) p = 95 %

684 µg/L

(CI: 607 - 770 µg/L)

1)       = Mortality> 20 % biologically significant

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

 

·   Water quality parameters as 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 dichromate as reference item once per month.
The EC50 of the reference item at 1.66 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.