Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05.01. - 08.01.2010
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 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Batch No.: DEBR006719
Purity (certified): 99.4 % (a/a) +/- 0.03 %
appearance: soldified melt, white
Expiry date: 2010-07-14
recommended storage: RT

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The test item concentrations were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end). Samples at start and end of the test were taken directly from the test replicates.

Test solutions

Vehicle:
no
Details on test solutions:
Stock solution: 200 mg/L, freshly prepared with dilution water
Dispersion treatment: 45 min magnetic stirring at 45°C and 1000 rpm

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
Culture medium: Nutrient medium Z according to Lüttge et al. (1994)
Preculture: A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted into each test concentration and the control.
Initial cell density: Approximately 2 - 5 x 103 cells/mL


Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
none

Test conditions

Hardness:
The medium had a nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
Nominal range: 21 – 22 °C
pH:
Test start: 8.0 - 8.1
Test end: 8.0 - 8.4
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Test concentrations: (nom.) 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L (factor: √10)
Test concentrations: 0.208 – 1.68 – 7.26 – 26.7 – 84.5 mg/L (factor: √10) (geometric mean measured)
Details on test conditions:
TEST METHOD

Procedure: static
Exposure duration: 72 h
Replicates: Three replicates for each concentration level, six for the control
Test container: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs.
Test volume: 25 mL
Dilution water: According to the guideline (OECD 201 medium)
This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.
Preculture: A four day old preculture was used as inoculum. Incubation was performed in 500 mL Erlenmeyer flasks with dilution water. For the start of the test the preculture was directly pipetted into each test concentration and the control.
Initial cell density: Approximately 2 - 5 x 103 cells/mL
Application: Application was carried out by adding appropriate volumes of the stock solution into each test concentration.
Temperature: Nominal range: 21 – 24 °C, controlled at  2 °C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity: 60 - 120 µE • m-2 • s-1
Light regime: 24 h/d light

TYPE AND FREQUENCY OF MEASUREMENT

Cell density and self fluorescence 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 each concentration level and the control. At the end it was measured from one pooled replicates of each concentration 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.

STATISTICS

The NOEC and LOEC were determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were done first. P-values for both Normality and Equal Variance Tests were 0.05. The a-value for ANOVA was a=0.05. Normality Test and Equal Variance Test failed when running a One Way Analysis of Variance for growth rates. Transformation (y = 10^y) was carried out with the yield values.

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 14.6 - 16.9
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
8.47 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 7.20 - 20.9
Details on results:
EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.
(see also "any other information on results incl. tables")

The nominal concentrations of 1.00 - 3.20 - 10.0 - 32.0 - 100mg/L corresponded to geometric mean measured test item concentrations of 0.208 – 1.68 – 7.26 – 26.7 – 84.5 mg/L. The concentrations of p-Chlorbenzaldehydand its metabolite chlorobenzoic acid were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of p-Chlorbenzaldehydat test start were in the range of 93 – 106 % of the nominal values. At the end of the test the measured concentrations of p-Chlorbenzaldehyd decreased whereas those of its metabolite chlorobenzoic acid increased correspondingly. The sum of p-Chlorbenzaldehydand its metabolite was calculated to be in the range of 4 – 83 % of the nominal values.
Results with reference substance (positive control):
Last test was performed on September 14 to 17, 2009, test concentrations were 0.25, 0.50 and 1.0 mg/L.
ErC50 = 0.600
EyC50 = 0.280

Any other information on results incl. tables

NOEC, LOEC and EC - values and 95 % Confidence Intervals of p-Chlorbenzaldehyd(0-72 h)

based on geometric mean measured test item concentrations [mg/L]

                       

 

Rate-related Inhibition

NOEC

1.68

LOEC

7.26

ErC10

8.63 (7.67 – 9.74)

ErC20

10.8 (9.71 – 11.9)

ErC50

15.8 (14.6 – 16.9)

 

Inhibition of Yield

NOEC

1.68

LOEC

7.26

EyC10

6.14 (2.75 – 18.9)

EyC20

6.92 (3.09 – 19.2)

EyC50

8.49 (7.20 – 20.8)

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 103cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of√10, nominal:1.00 - 3.20 - 10.0 - 32.0 - 100mg/L corresponding to geometric mean measured test item concentrations of 0.208 – 1.68 – 7.26 – 26.7 – 84.5 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

p-Chlorbenzaldehydwas found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (geometric mean measuredtest item concentrations): The EC50 -values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 15.8 (14.6 – 16.9) and 8.49 (7.20 – 20.8) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of p-Chlorobenzaldehyd.