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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 21 MAR 2011 to 25 MAR 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study compliant to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to German "Chemikaliengesetz", EC and OECD Guideline principles

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: C-Wax Type OP
Chemical Name: Fatty acids, carnauba wax, butyleneglycol esters
Content: 100%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The limit loading level and control were analytically verified in the fresh media (0 h) and old media (72 h) via analysis of total organic carbon (TOC, according to DIN EN 1484). Separate replicates for the test item analysis were prepared without algae and incubated under test conditions.

Test solutions

Vehicle:
no
Details on test solutions:
One water accommodated fraction (WAF) of 100 mg/L was prepared with dilution water one day prior to application. The WAF was shaken with 20 rpm for 24 h at room temperature. After a separation phase of 1 h the WAF was taken from the homogeneous liquid phase.
Per definition of the WAF all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The water accomodated fraction (WAF) was tested in a limit test.

The WAF is defined as the aqueous medium containing only the fraction of a multi-component test item that is dissolved and / or present as a stable dispersion/emulsion under test conditions in the test medium, acc. to OECD Guidance Document, No. 23.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): four day old preculture.
- Preculture: Incubation was performed in 500 mL Erlenmeyer flasks with dilution water.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE·mE(-2)·sE(-1) for 24 h per day.
- Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994).

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None. Observations were carried out during exposure at the beginning of the experiment and after 24, 48 and 72 hours.

Test conditions

Hardness:
0.24 mmol Ca+Mg/L for the dilution water
Test temperature:
21-22 (mean: 21.5) deg C
pH:
Start: 7.74 (test solution), 8.11 (control)
End, 72 h: 8.08 (test solution), 8.20 (control)
Nominal and measured concentrations:
The original WAF was tested in a limit test (100 mg/L nominal test item concentration).
Measured concentrations (DOC-analysis) were close to the limit of detection, as the test item is insoluble in water. Therefore, all concentrations are nominal concentations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile erlenmeyer flasks, volume: 250 mL sealed with cotton wool plugs.
- Initial cells density: nominal 2-5 x 10E(3) cells/mL - current 4 x 10E(3) cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM / dilution water
- Standard medium used: yes (OECD 201 medium)
- Source/preparation of dilution water: dilution water according to guidelines


OTHER TEST CONDITIONS
- Photoperiod: 24 h/d
- Light intensity and quality: 60-120 µE x mE(-2) x sE(-1)
- Application was carried out by adding appropriate volumes of the water accommodated fraction to the test replicates.
- Temperature: Nominal range: 21 - 24 deg C, controlled at +/- 2 deg C
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

TYPE AND FREQUENCY OF MEASUREMENT:
Cell density was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. The pH-value at the beginning of the test was measured out of one additional replicate from the limit loading level and the control. At the end it was measured from one pooled replicates of the limit loading level and the control. The room temperature was measured continuously by a hygro-thermograph. Light intensity was measured prior to test start. Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

CALCULATIONS
All calculations were based on the fluorescence values of each replicate.
Cell density out of fluorescence values:
The fluorescence values were related to cell density values according to a calibration curve.
x = 5.6 x Fq + 429
x = cell density [cells/mL]
Fq = mean fluorescence
The growth rate, yield, the rate related inhibition and the inhibition of yield after 72 h were calculated from the cell density values according to the guideline.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate p.a.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities. All effect values are given based on nominal loading of the test item (water accomodated fractions).
For details see table 1 in section "any other information on results including tables".
Results with reference substance (positive control):
The toxicity of potassium dichromate (SIGMA, batch number MKBC1858, purity 99.9 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 h from September 20 to 23, 2010. The reference item toxicity is in the valid range following test facility SOPS.

Effect values [mg/L], 0-72h

EC50 (growth rate) 0.589
95 % confidence interval 0.535 - 0.652
Valid Range (average +/- 3 x SD): 0.738 +/- 0.437

EC50 (yield) 0.263
95 % confidence interval 0.252 - 0.274
Valid Range (average +/- 3 x SD): 0.328 +/- 0.146
Reported statistics and error estimates:
NOEL/LOEL was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT's Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO.WILK-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test were 0.05.
The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

EL-values and statistical analyses:
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (limit test design).

SOFTWARE
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if
calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.

Any other information on results incl. tables

Table 1:

Evaluation after 72 h

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item loading

Replicate

Growth rate

Rate-related inhibition

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

100

1

 

1.58

-3.84

 

465937

-19.1

2

 

1.59

-4.11

 

471705

-20.6

3

 

1.60

-4.77

 

486209

-24.3

4

 

1.57

-2.77

 

443509

-13.4

5

 

1.67

-9.20

 

596501

-52.5

6

 

1.64

-7.55

 

552765

-41.3

Mean

(+)*

1.61

-5.37

(+)*

502771

-28.5

Control

1

 

1.55

 

 

421389

 

2

 

1.51

 

 

369141

 

3

 

1.53

 

 

391737

 

4

 

1.50

 

 

365389

 

5

 

1.50

 

 

365501

 

6

 

1.56

 

 

433653

 

Mean

 

1.53

 

 

391135

 

negative inhibitions = increase of growth

*) significance caused by increase of growth

Table 2:   TOC Content of the Test Media at Test Start and Test End

 

Nominal test item loading
[mg/L]

TOC[mg/L]

TOC[mg/L]

Test start (without alga), 0 h

Test end (without alga), 72 h

Parallel determination

Mean

Parallel determination

Mean

100

4.7

4.7

4.7

3.8

3.8

3.8

Control

< 2.0

< 2.0

< 2.0

< 2.0

< 2.0

< 2.0

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a growth inhibition test on freshwater green algae according to OECD 201 no inhibiting effects on biomass growth and specific growth rate were found for the water accomodated fraction of the submission substance. The EC50-values for inhibition of biomass growth (EbC50) and specific growth rate (ErC50) after 72 h were > 100 mg/L nominal concentration.
Executive summary:

The toxicity of C-Wax Type OP to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 and compliant to GLP (reliability category 1) at DR.U.NOACK-LABORATORIEN in 31157 Sarstedt, Germany from March 21 to 25, 2011 with the definitive exposure phase from March 22 to 25, 2011. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of 4 x 10 E(3) cells/mL. Based on a preliminary range finding test, a limit test with a water accommodated fraction (WAF)* was carried out. The water accommodated fraction was prepared with a nominal loading of 100 mg/L test item C-Wax Type OP/L with dilution water one day prior to application. The WAF was shaken with 20 rpm for 24 h at room temperature. After a separation phase of 1 h the WAF was taken from the homogeneous liquid phase. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The concentration of C-Wax Type OP was determined at the beginning and end of the test via TOC-analysis at the limit loading level and control. Due to the insolubility of the test item in water, all effect values are given based on the nominal loading of the test item (water accomodated fraction according to OECD Guidance Document, No. 23 ):

NOEC (72h, growth rate and yield) = 100 mg/L;

EC10 (72h, growth rate and yield) > 100 mg/L;

EC50 (72h, growth rate and yield) >100 mg/L.