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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-22 2009-09-25 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to relevant guidelines and compliant to GLP. The results are plausible and well documented.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
According to § 19b German chemical act and directive 88/320/EEC
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
yes
Details on sampling:
DETERMINATION OF THE TEST ITEM
The concentration of the test item were analytically determined from all test concentrations and the control after 0 h (test start and 72 h (test end). Separate samples for the test start (prepared without algae) were provided. Samples at the end of the test were taken directly from the test replicates.

PREPARATION OF SAMPLES
The test item concentrations 3.2 mg/L to 100 mg/L were diluted with mobile phase prior to analysis. The concentration of 1.0 mg/L and the control were analysed directly. At test end, all samples were filtered.

SAMPLE STORAGE
All samples were stored at 6 ± 2 °C until start of analysis if necessary.
Vehicle:
no
Details on test solutions:
Stock solution of the test item: 200 mg/L, freshly prepared with dilution water.
Dispersion treatment: Agitation
Test concentrations: 1.0 - 3.2 - 10 - 32 - 100 mg/L (factor √10)
(nominal)
Test concentrations 0.696 – 2.25 – 7.41 – 26.0 – 75.4 mg/L
(geometric mean measured)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
Desmodesmus subspicatus CHODAT SAG 86.81 (formerly Scenedesmus subspicatus CHODAT);
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
Cultivation at test facility: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35-70 µE•mE(-2)•sE(-1) for 24 h per day
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
4-7 days to test for reversibility of growth inhibition.
Hardness:
0.24 mmol Ca and Mg /L
Test temperature:
min: 21 °C
max: 23 °C
mean: 22 °C
pH:
8.1 +/- 1.2
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Test concentrations (nominal): 1.0 - 3.2 - 10 - 32 - 100 mg/L (factor √10)
Test concentrations (geometric mean measured): 0.696 – 2.25 – 7.41 – 26.0 – 75.4 mg/L
The concentrations of o-Chlorbenzaldehyd and its metabolite chlorobenzoic acid were analysed at all concentration levels after 0 h (without algae) and 72 h (same algae density as test replicates) via HPLC analysis. The measured concentrations of o-Chlorbenzaldehyd at test start were in the range of 78 - 98 % of the nominal values. At the end of the test the measured concentrations of o-Chlorbenzaldehyd decreased whereas those of its metabolite chlorobenzoic acid increased correspondingly. Therefore, the sum of o-Chlorbenzaldehyd and its metabolite was calculated to be in the range of 53 — 68 % of the nominal values. All effect values are given based on geometric mean measured concentrations of o-Chlorbenzaldehyd.


Details on test conditions:
CONTROL: Six replicates (without test item) were tested under the same test conditions as the test replicates.
SELF FLUORESCENCE: No self fluorescence has been found up to the concentration of 75.8 mg/L.
TEST METHOD: Static procedure
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: 100 mL

Dilution water: According to the guideline (OECD-medium);
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 to the test and control replicates to receive an initial cell concentration of approximately 2 – 5 x 10E(3) cells/mL in the replicates.

Application Application was carried out by adding appropriate volumes of the stock solution to the test replicates.
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 • mE(-2) • sE(-1)
Light regime 24 h/d light

Recovery of algae: After 72 h algae were transferred from geometric mean measured test concentrations 7.41 – 75.4 mg/L and control into fresh untreated medium (0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further
4 – 7 d to determine whether the effect of the item is reversible.

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 each concentration level and the control. At the end it was measured from one pool of all 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.

COURSE OF THE STUDY
- Preliminary range finding test (non GLP)
- Incubation of the preculture
- Preparation of the stock solution
- Application
- Incubation
- Determination of cell density after 0, 24, 48 and 72 h
- Determination of the test item after 0 and 72 h
- Evaluation

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 = 5.9 • Fq – 1612
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:
REFERENCE ITEM: Potassium dichromate p.a. (Sigma) CAS No. 7778-50-9; Purity: ≥ 99.5 %; Batch number: 06719MH; Test concentrations: 0.28 - 0.51 - 0.90 - 1.6 mg/L; Experimental date: September 22 to 25, 2008
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
7.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.84 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 1.79 - 3.88
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
5.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 3.84 - 6.37
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 13.8 - 20.9
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
7.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 2.58 - 3.78
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
3.46 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 2.95 - 4.25
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.67 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 3.88 - 5.57
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 geometric mean exposure concentrations of the test item.
Environmental conditions pH-value, measured at 0 and 72 h, and room temperature (measured continuously) were determined to be within the acceptable limits.
Results with reference substance (positive control):
The toxicity of potassium dichromate to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 h from September 22 to 25, 2008. The reference item toxicity is in the valid range following NOACK-LAB SOPS.

EC50 Values of the Reference Item based on nominal concentrations [mg/L], (0-72 h)

Rate-related inhibition
ErC50 (Current Study): 0.69 Valid Range (average ± 3 x SD): 0.828 ± 0.399
95 % confidence interval 0.64 – 0.74

Yield inhibition
EyC50 (Current Study): 0.31 Valid Range (average ± 3 x SD): 0.352 ± 0.143
95 % confidence interval 0.30 – 0.32


Reported statistics and error estimates:
NOEC, LOEC and statistical analyses:
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. The Equal Variance Tests of growth rate data failed. The data were transformed with Graph Pad Prism function Y = sqrt (Y).

EC-values and statistical analyses:
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.

  Recovery of Alga Cells

 After 72 h of exposure, 0.5 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from geometric mean measured test item concentrations 7.41 – 75.4 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 - 7 days under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 75.4 mg/L.

Range Finding Test (non GLP)

 

  Results of the Range Finding Test (0 – 72 h)

Nominal
Test Item Concentration
[mg/L]

Specific Growth Rate Inhibition

[%]

Yield Inhibition


[%]

100

100

100

10

8

38

1

-3

-18

                  negative inhibitions = increase of growth

Validity criteria fulfilled:
yes
Remarks:
The study meets the validity criteria of the guideline: - The cell growth increased 181-fold after 72 h in the control (required: 16-fold). - The temperature during the test was in the range of 21-24 °C. The difference from the mean was within ± 2 °C: mi
Conclusions:
In a 72 h growth inhibition test on freshwater green alga Desmodesmus subspicatus according to relevant guideline and performed complient to GLP (reliability category 1) 2-Chlorobenzaldehyde was found to inhibit the growth after 72 h with the following effect values (geometric mean exposure concentrations): The EC50-values with 95 % confidence intervals for specific growth rate (ErC50) and yield (EyC50) after 72 h were 16.8 (13.8 – 20.9) and 4.67 (3.88 – 5.57) mg/L, respectively. All effect levels are given based on geometric mean measured concentrations of 2-Chlorobenzaldehyde.
Executive summary:

The toxicity of 2-chlorobenzaldehyde to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201. 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 approximately 2 – 5 x 103cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 1.0 - 3.2 - 10 - 32 - 100 mg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of 2-chlorobenzaldehyde and its metabolite chlorobenzoic acid were analysed at all concentration levels after 0 h (without algae) and 72 h (same algae density as test replicates) via HPLC analysis. The measured concentrations of 2-chlorobenzaldehyde at test start were in the range of 78 - 98 % of the nominal values. At the end of the test the measured concentrations of 2-chlorobenzaldehyde decreased whereas those of its metabolite chlorobenzoic acid increased correspondingly. Therefore, the sum of 2-chlorobenzaldehyde and its metabolite was calculated to be in the range of 53 – 68 % of the nominal values. All effect values are given based on geometric mean measured concentrations of 2-chlorobenzaldehyde.

 

                  NOEC, LOEC, EC10-,EC20-and EC50- values and 95 % Confidence Intervals of
2-chlorobenzaldehyde (0-72 h) based on geometric mean measured concentrations of the test item [mg/L]

                               

 

Rate-related inhibition

NOEC

2.25

LOEC

7.41

ErC10

2.84 (1.79 – 3.88)

ErC20

5.04 (3.84 – 6.37)

ErC50

16.8 (13.8 – 20.9)

 

Inhibition of yield

NOEC

2.25

LOEC

7.41

EyC10

3.02 (2.58 – 3.78)

EyC20

3.46 (2.95 – 4.25)

EyC50

4.67 (3.88 – 5.57)

 

After 72 h algae were transferred from geometric mean measured concentrations 7.41 – 75.4 mg/L and control to fresh untreated medium and allowed to grow for further 4 - 7 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 75.4 mg/L (highest test concentration).

Description of key information

In a 72 h growth inhibition test on freshwater green alga Desmodesmus subspicatus according to relevant guideline and performed complient to GLP (key study, reliability category 1) 2-Chlorobenzaldehyd was found to inhibit the growth after 72 h with the following effect values (geometric mean measured exposure concentrations, growth reduction, mg/L): NOEC = 2.25; LOEC = 7.41; EC10 = 2.84; EC20 = 3.46; EC50 = 16.8.

Key value for chemical safety assessment

EC50 for freshwater algae:
16.8 mg/L
EC10 or NOEC for freshwater algae:
2.84 mg/L

Additional information

In a 72 h growth inhibition test on freshwater green alga Desmodesmus subspicatus according to relevant guideline and performed complient to GLP (reliability category 1) 2-Chlorobenzaldehyd was found to inhibit the growth after 72 h with the following effect values (geometric mean exposure concentrations): The EC50-values with 95 % confidence intervals for specific growth rate (ErC50) and yield (EyC50) after 72 h were 16.8 (13.8 – 20.9) and 4.67 (3.88 – 5.57), respectively. All effect levels are based on geometric mean measured concentrations of 2 -Chlorobenzaldehyde.

Besides this study, experimental data are only available for Chlorella. In this non-guideline study, enzymatic activity mirrowed viability. Thus, a 15 minute EC50 of 122 mg/L had been determined. However firstly, viability is a less sensitive endpoint than the growth rate and secondly the incubation time was 15 minutes instead of 72 hours according to OECD 201.

QSAR results are not assignable due to the missing information on validation criteria. The Danish EPA-QSAR-Database result on Selenastrum confirms for 2-chlorobenzaldehyde to be within the model domain and delivers an estimated EC50 (acute) for Selenastrum of 6.1 mg/L. Being only lower by a factor of 2, this value is fitting quite well with the experimental data of the key study on Desmodesmus subspicatus.