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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-21 to 2012-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study; EU method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).

Test solutions

Vehicle:
no
Details on test solutions:
Dosage of Test Item:
The test medium of the highest test concentration of nominal 10000 mg test item/L was prepared by dissolving 7001.5 mg test item into 700.15 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
SSpecies: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
22 to 23°C
pH:
8.1 at test start and
8.0 to 8.7 at test end
Nominal and measured concentrations:
Test Concentrations:
10000, 5000, 2500, 1250 and 625 mg test item/L and a control.
Details on test conditions:
Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was:
22 to 23 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was:
8.1 at test start and
8.0 to 8.7 at test end
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity:
6103 lux (range: 5680 to 6680 lux)

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8 996 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL:8211-10048
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2 871 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 2283 - 3378
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 625 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2 814 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL:2495 - 3177
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
769 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL:568- 959
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 625 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
The 72-hour ErC50 value was calculated to be 8996 mg test item/L and the 72-hour EbC50 was calculated to be 2814 mg test item/L. The 72-hour NOErC and the 72-hour NOEbC were determined to be lower than 625 mg test item/L and the associated 72-hour LOEC for both parameters is 625 mg test item/L at most.
Reported statistics and error estimates:
Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by the Welch t-test (growth rate) and the Williams t-test (yield), respectively.

The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Any other information on results incl. tables

Biological Results

Yield y and Percentage Inhibition of y during the Test Period

Nominal concentration Yields y [104cells/mL] and % inhibition of y
24 hours   48 hours   72 hours
[mg test item/L] y %   y %   y %  
                   
                   
Control 3.871 - 29.496 - 109.217 -  
625 5.086 -31.4   28.471 3.5   98.246 10.0 *
1250 3.795 2.0   23.232 21.2 * 82.074 24.9 *
2500 2.352 39.2 * 17.765 39.8 * 66.130 39.5 *
5000 1.289 66.7 * 7.819 73.5 * 31.887 70.8 *
10000 0.986 74.5 * 2.504 91.5 * 5.086 95.3 *
                   

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* mean value significantly different from the control(tested with Williams t-test,a = 0.05, one-sided)

Growth Rates µ and Percentage Inhibition of µ during the Test Period

                   
Nominal concentration Growth rates µ [1/day] and % inhibition of µ
0 - 24 hours   0 - 48 hours   0 - 72 hours
[mg test item/L] µ %   µ %   µ %  
                   
                   
Control 2.161 - 2.046 - 1.796 -  
625 2.412 -11.6   2.030 0.8   1.762 1.9 *
1250 2.148 0.6   1.930 5.7 * 1.701 5.3 *
2500 1.739 19.5 * 1.799 12.1 * 1.630 9.2 *
5000 1.270 41.2 * 1.406 31.3 * 1.390 22.6 *
10000 1.079 50.1 * 0.894 56.3 * 0.789 56.1 *
                   

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control
(tested with Welch t-test,a = 0.05, one-sided)

 

Analytical Results

Determination of the Test Item:

Based on the results of GC measurements the concentration of the test item was determined using a calibration curve.

Calibration:

0.5 to 10 mg test item/L

Linearity of Response:

Correlation of peak area of different standard solutions with their corresponding concentrations, using a linear regression.

Regression Coefficient:

At least 0.9986


Calibration Curve:

y = 38846 * x - 2788

Limit of Detection:

0.048 mg test item/L

Limit of Quantification:

250 mg test item/L

mean recovery rate: 102 % (n = 5; RSD 2 %)

Mean Recovery in the Fortified Samples:


99 % (n = 17, RSD 4 %)

Mean Recovery in the Test Samples:


After 0 h:       108 % (n = 10, RSD 13 %)

After 72 h:     107 % (n = 10, RSD 5 %)

 

 

Table3. Summary of Analytical Results

       
sample description % of RSD
[mg test item/L] nominal1 [%] n
       
control n.a. n.a. 2
625 100 3 4
1250 101 14 4
2500 116 9 4
5000 112 5 4
10000 110 5 4
       

1mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

 

 

 

The limit of detection and the limit of the quantification of the analytical method were 0.048 and 250 mg test item/L, respectively.

At the start of the test 108 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 107 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 108 % of nominal. Therefore, all reported results refer to nominal concentrations.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The quantification of the test item was performed using gas chromatography (GC-MS-method). The limit of detection and the limit of the quantification of the analytical method were 0.048 and 250 mg test item/L, respectively.
At the start of the test 108 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 107 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 108 % of nominal. Therefore, all reported results refer to nominal concentrations.


The influence of Tetraethyleneglycoldimethylether on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC50 value was calculated to be 8996 mg test item/L and the 72-hour EbC50 was calculated to be 2814 mg test item/L. The 72-hour NOErC and the 72-hour NOEbC were determined to be lower than 625 mg test item/L and the associated 72-hour LOEC for both parameters is 625 mg test item/L at most.
Executive summary:

The toxicity of the test item to Pseudokirchneriella subcapitata in an algal growth inhibition test was determined based on the procedures indicated by the following internationally accepted method:

- OECD Guideline No 201

- EU Method C.3

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. Exponentially growing cultures of this unicellular green algal species were exposed to varios concentrations of the test item under defined conditions.The inhibition growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

Test Concentrations: 10000, 5000, 2500, 1250 and 625 mg test item/L and a control.

At the start of the test 108 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 107 % of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 108 % of nominal. Therefore, all reported results refer to nominal concentrations.

T The influence of Tetraethyleneglycoldimethylether on the growth of the freshwater green algaePseudokirchneriella subcapitatawas assessed in a static dose-response test.The 72-hour ErC50value was calculated to be8996 mg test item/L and the 72-hour EbC50was calculated to be2814 mg test item/L. The 72-hour NOErC and the 72-hour NOEbC were determined to be lower than 625 mg test item/L and the associated 72-hour LOEC for both parameters is 625 mg test item/L at most.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.