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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-08-12 to 2009-09-08
Reliability:
1 (reliable without restriction)

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
Details on test material:
- Name of test material (as cited in study report): HYEQS
- Chemical name: Quaternary ammonium compounds, C12-18-alkyl (hydroxyethyl) dimethyl, chlorides
- Physical state: Solid, white granules
- Analytical purity: 98.8 %
- Purity test date: 2009-06-10
- Lot/batch No.: DEG4052477
- Expiration date of the lot/batch: 2011-03-01
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels of the test item and the control were analytically verified in the fresh media (0 hours) and old media (24 hours) via LC-MS/MS once every 7days.
- Sampling method: For the fresh media samples of the freshly prepared test solutions were taken in duplicate. For the old media samples were taken in duplicate directly from the test vessels. The samples of fresh and old media were stabilized by diluting 1:2 with acetonitrile + 0.5 % trifluoro acetic acid. One sample per concentration level and control was analysed.
The quat may sorb to the surface of the glass vessel and / or the test organisms and may reduce the analytical recovery below the acceptable level. Therefore additionally prepared replicates with test item but without test animals were used in addition to demonstrate the amount of sorption to the glass surface. The additionally prepared replicates without test organisms were incubated under test conditions until analysis.

- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C after sample preparation, if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared. Dispersion treatment was agitation
- Eluate: Dilution water
- Differential loading: 12.5 - 25.0 - 50.0 - 100.0 - 200.0 µg/L
- Controls: 10 replicates of dilutionwater 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
Test Item
Concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
August 12th August 13th August 19th August 20th August 26th August 27th
200 214 228 201 177 185 194
Control 177 217 177 177 174 185
Test temperature:
Temperature [°C]
Nominal
Test Item
Concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
August 12th August 13th August 19th August 20th August 26th August 27th
200 21.2 21.2 21.4 21.9 21.2 21.8
Control 21.1 21.2 21.4 22.0 21.6 22.0
pH:
pH-Values
Nominal
Test Item
Concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
August 12th August 13th August 19th August 20th August 26th August 27th
200 7.59 7.84 7.88 7.50 7.85 7.79
Control 7.61 7.69 8.06 7.41 7.98 7.56
Dissolved oxygen:
Dissolved Oxygen Concentration [mg/L]
Nominal
Test Item
Concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
August 12th August 13th August 19th August 20th August 26th August 27th
200 8.24 8.93 7.57 7.32 8.05 9.92
Control 8.04 7.83 8.02 6.84 8.40 9.39
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
Immobilization Rates of the non GLP Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[µg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1. 2. MV 1. 2. MV
1000 100 100 100 100 100 100
100 0 0 0 0 0 0
10 0 0 0 0 0 0
1 0 0 0 n.d.
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.:
100 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality of parent animals: According to the guideline adult mortality of > 20 % is seen to be biologically substantial. At the concentration levels of 12.5, 50.0 and 100 µg/L adult mortality of 10 % was observed. At the control and the nominal test item concentration 25.0 µg/L all daphnids survived until the end of the study. An EC50 for the adult mortality after 21 days could not be estimated because adult mortality ≥ 50 % was not observed within the tested concentration range. The adult mortality came to a maximum of 30 % at the concentration level 200 µg/L.
- No. of offspring produced per day per female: Five broods were observed in the test period at all parent animals of the control and the test groups. The average number of juveniles per parent in the control group was 144 after 21 days. The reproductive output was statistically significant reduced in comparison to the control at the nominal test item concentration 200 µg/L. The other concentration levels 12.5 to 100 µg/L were not statistically significant reduced when compared to the control (One Way Analysis of Variance, DUNNETT’S method, p = 0.05). An EC50 for the reproductive output could not be estimated, because a reduction ≥ 50 % was not observed within the tested concentration range.
- 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 in the replicates producing juveniles of all concentration levels and the control was between day 7 and day 9
- Other biological observations: Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the tested concentration levels 12.5 to 200 µg/L. At the control no stillborn juveniles or aborted eggs were observed.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.50 (CI 1.39 - 1.63)
Reported statistics and error estimates:
The NOEC and LOEC for the reproductive output as the most sensitive effect were determined directly from the reduction of the reproductive output. Significant deviations were determined in comparison to control using statistical standard procedures as Normality Test, Equal Variance Test and One Way Analysis of Variance (ANOVA).
Prior to running a One Way Analysis of Variance 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 and the body length One Way Analysis of Variance, DUNNETT’S method, p = 0.05 was used. Statistical evaluation of the intrinsic rates of natural increase was carried out using One Way Analysis of Variance, p = 0.05. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The EC50-value of the reference test were calculated using sigmoidal dose-response regression. The 95 % confidence limits of the EC50 were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.
EC50-values for the reproductive output and the adult mortality could not be estimated, because effects ≥ 50 % were 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

Test

Item

Conc.

[µg/L]

No.

of

Parents

prod. Juv.

Mean No. of Juveniles

per Parent

producing Juveniles

Comparison

versus

Control

Number of Juveniles in Replicate No.

Total

No.

CV

Red.

Stat.

  1

  2

  3

  4

  5

  6

  7

  8

  9

   10

å

N

MV ± SD

[%]

[%]

200

116

114

--

100

109

--

112

--

125

154

  830

  7

119 ±

17

15

17

yes

100

154

163

135

145

132

160

156

154

--

133

1332

  9

148 ±

12

  8

 -3

no

  50.0

182

151

143

168

137

163

151

153

178

--

1426

  9

158 ±

15

10

-10

no

  25.0

136

136

137

160

146

155

130

122

180

166

1468

10

147 ±

18

12

 -2

no

  12.5

145

151

146

--

167

135

157

174

144

156

1375

  9

153 ±

12

  8

 -6

no

Control

138

148

137

141

146

148

138

137

149

155

1437

10

144 ±

  6

  4

First Appearance of Living Juveniles in the Individual Groups

Nominal

Test Item

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

200

8

8

--

8

8

--

7

--

8

7

7.7

100

7

8

9

8

7

7

7

9

--

8

7.8

    50.0

8

8

8

8

8

8

8

8

7

--

7.9

    25.0

8

8

8

8

8

8

8

8

7

8

7.9

    12.5

8

8

7

--

8

8

7

8

8

8

7.8

Control

8

8

8

8

8

8

8

8

8

8

8.0

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

                 (n = 10)

Nominal Test Item Concentration

Adult Mortality [%]

[µg/L]

7 days

14 days

21 days

200

0

10

 30*

100

0

  0

10

    50.0

0

  0

10

    25.0

0

  0

  0

    12.5

0

  0

10

Control

0

  0

  0

Total Body Length of the Parental Daphnids

Nominal

Test Item

Concentration

[µg/L]

Total Length of the Parent Animals

[mm]

MV

Red.

N

Replicate No.

1

2

3

4

5

6

7

8

9

10

[mm]

[%]

200

5.00

5.00

4.75

--

5.00

--

5.25

--

5.00

4.50

4.931)

 9

  7

100

5.00

5.25

4.75

4.50

5.00

5.00

5.50

5.00

--

5.00

5.001)

 8

  9

    50.0

5.75

5.50

5.25

5.75

6.00

5.50

5.25

5.50

5.50

--

5.56

-2

  9

    25.0

5.00

5.00

5.25

5.50

5.75

6.00

5.50

5.25

5.75

5.75

5.48

-1

10

    12.5

5.75

5.50

5.75

--

5.50

5.50

6.00

5.25

5.75

6.00

5.67

-4

  9

Control

5.50

5.50

5.25

5.75

5.25

5.50

5.25

5.25

5.50

5.50

5.43

10

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The Lowest Observed Effect Concentration (LOECReproduction) and the No Observed Effect Concentration (NOECReproduction) for the reproductive output as the most sensitive effect were determined directly from reduction of the reproductive output. EC50- values for the reproductive output and the adult mortality could not be estimated because effects ≥ 50 % were not observed.
All effect values given are based on the nominal concentrations of the test item HYEQS.

NOECReproduction : 100 µg/L
LOECReproduction : 200 µg/L

NOECAdult Mortality : 100 µg/L
LOECAdult Mortality : 200 µg/L
Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item HYEQS (Hydroxylethylquad Solid, batch No. DEG4052477) was conducted according to OECD 211 (1998) from 2009-08-12 to 2009-09-08 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1,
D-31157
, Germany .

The test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At test start they were 2 to 24 hours old. The study was carried out undersemi-static conditions with daily renewal of the test solutions. The 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 time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of HYEQS were selected as follows: 12.5 - 25.0 - 50.0 - 100 - 200 µg/L.

Cationic surfactants are difficult to test as they exhibit multiple sorption mechanisms including classical van der Waals, ionic interaction and ion exchange. Cationic surfactants may sorb to negatively charged surfaces like glass and the surface of the daphnids and algae but there is currently no mechanistic model to estimate the degree of sorption. Sorption may lead to low analytical recoveries of the test item during the test and it is therefore important to address sorption effects either to the daphnids and/or the glass surface of the test vessels.

The concentrations of HYEQS (C12-Fraction and C14-Fraction), active ingredients of the test item HYEQS were analytically verified via LC-MS/MS of samples on days 0, 7, 14 (fresh media, 0 hours) and on days 1, 8, 15 (old media, 24 hours) at all concentration levels. Details of the analytical method are presented in part 10. The recoveries in the fresh media (0 hours) were determined to be 99 to 139 % for the active ingredient HYEQS (C12-Fraction) and 98 to 141 % for the active ingredient HYEQS (C14-Fraction).

The recoveries in the old media (24 hours) determined from samples taken directly from the test vessels, containing daphnids and algae, were in the range of 100 to 122 % for the active ingredient HYEQS (C12-Fraction) and 62 to 95 % for the active ingredient HYEQS (C14-Fraction).

Additional replicates were prepared and incubated without daphnids and algae and gave recoveries in the range of 116 to 129 % for the active ingredient HYEQS (C12-Fraction) and 110 to 144 % for the active ingredient HYEQS (C14-Fraction) which were comparable to the recoveries of the old media in the test vessels with daphnids and algae.

As the recoveries in the old media with and without daphnids were comparable and only a negligible decrease of the recoveries in the old media after 24 hours in comparison to those in the fresh media was observed, it can be concluded that sorption of the test item to the daphnids and algae can be neglected for this Hydroxylethylquat solid.


Based on these facts a correction of the nominal concentrations is not justified as the test item is not sorbed or degraded to an unacceptable degree during the test and was therefore available for exposure of the daphnids in the test medium. Nominal concentrations can be used for establishing the effect values.

All effect values given are based on the nominal concentrations of the test item HYEQS.

·   The average number of juveniles per parent in the control group was 144 after 21 days. The reproductive output was statistically significant reduced in comparison to the control at the nominal test item concentration 200 µg/L. The other concentration levels 12.5 to 100 µg/L were not statistically significant reduced when compared to the control (One Way Analysis of Variance, Dunnett’smethod, p = 0.05).
An EC50-value for the reduction of the reproductive output could not be estimated, because a reduction of the reproductive output ≥ 50 % was not observed within the tested concentration range
.

·   The coefficient of variation of the number of living offspring produced per parent was 4 % in the control.

·   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 mean IR of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance (p = 0.05). There was no statistically significant difference at any of the tested concentration levels 12.5 to 200 µg/L when compared to the control.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was lower than 1 % at the tested concentration levels 12.5 to 200 µg/L . In 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 and by all parent animals of the control and the nominal test item concentrations of 12.5 to 200 µg/L.

·   The Lowest Observed Effect Concentration (LOECReproduction) and the No Observed Effect Concentration (NOECReproduction) after 21 days based on the reproductive output as the most sensitive effect are summarized in Table 1.

Test Item Related Effects on Reproduction (NOEC and LOEC)

Effects

HYEQS

Nominal Test Item Concentration [µg/L]

Control

12.5

25.0

50.0

100

200

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

144 ± 6

153 ± 12

147 ± 18

158 ± 15

148 ± 12

119 ± 171)

Mean Intrinsic Rates of Natural Increase

0.51

0.53

0.52

0.53

0.53

0.53

Appearance of First Brood [Mean Day]

8.0

7.8

7.9

7.9

7.8

7.7

NOECReproduction

100 µg/L

LOECReproduction

200 µg/L

SD = Standard deviation

1)    = Statistical significance (One Way Analysis of Variance, Dunnett’s method, p = 0.05)

A summary of test item related effects concerning the adult mortality (NOECAdult Mortality and LOECAdult Mortality) and growth (dry weight, body length) is given in Table 2.

·   The test item induced biologically substantial adult mortality of 30 % at the nominal test item concentration 200 µg/L after 21 days. According to the guideline adult mortality of > 20 % is seen to be biologically substantial. At the concentration levels of 12.5, 50.0 and 100 µg/L adult mortality of 10 % was observed. Atthe control and the nominal test item concentration 25.0 µg/L all parental daphnids survived until the end of the study. An EC50for the adult mortality after 21 days could not be estimated because adult mortality ≥ 50 % was not observed within the tested concentration range.

·   The mean body lengths of the surviving parental daphnids at the nominal test item concentrations 12.5 to 200 µg/L were determined to be in the range of 4.93 to 5.67 mm per daphnid and 5.43 mm per daphnid at the control group, respectively (Table 10). The body length was statistically significantly reduced to a mean value of 5.00 mm per daphnid at concentration level 100 µg/L and a mean value of 4.93 mm per daphnid at concentration level 200 µg/L (One Way Analysis of Variance, p = 0.05). The statistical significance is regarded to be biologically not relevant, because the reduction was lower than 10 %, which is seen to be within an acceptable variation range for this parameter.