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Ecotoxicological information

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:
25 March to 24 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
Swiss GLP Compliance Programme (inspected on 22-26 March and 12-16 April 2010 / signed on 11 January 2011)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Chimex / 0141653- Expiration date of the lot/batch: 30 June 2011- Purity test date: 28 September 2010STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature at about 20 °C, in the dark
Analytical monitoring:
yes
Details on sampling:
Analysis of the Test Item ConcentrationsFor measurement of the actual concentrations of the test item, duplicate samples (without algae) were taken from each treatment at the start of the test. At the end of the test, samples (with algae) were taken in triplicate from the control and the lower test concentrations of 0.023 to 0.11 mg/L and in duplicate from the two highest test concentrations of 0.23 and 0.50 mg/L.For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. A volume of up to 500 mL per sample was necessary for analytical purposes.All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.The concentrations of the test item Chimexane CL were determined in the samples of the three highest test concentrations of nominal 0.11 to 0.50 mg/L and in the control. One sample was analyzed per sampling time. The samples from the lower nominal test concentrations of 0.023 and 0.050 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION- Method: A stock solution with a nominal concentration of 50 mg/L was prepared by dissolving 50.14 mg of the test item in 1000 mL of test water stirring intensely for 5 minutes at room temperature. Adequate volumes of this stock solution were diluted with test water to prepare the test media of all test item concentrations. The test media were prepared just before the start of the test.- Controls: Test water without test item
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM - Strain: Strain No. 61.81 SAG - Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany - Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
0.24 mmol/L (= 24 mg/L as CaCO3)
Test temperature:
22 °C
pH:
At the start of the test, the pH measured in the treatments was between 8.2 and 8.4. At the end of the test, pH values of 8.5 to 9.8 were measured.
Dissolved oxygen:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentrations of the test item of 0.023, 0.050, 0.11, 0.23 and 0.50 mg/L (corresponding to 0.0069, 0.015, 0.033, 0.069 and 0.15 mg hydroxyethyl oleyl dimonium chloride/L corrected for water content)
Details on test conditions:
TEST SYSTEM - Test vessel: 50 mL Erlenmeyer flasks - Type: Test flask was covered with a glass dish - During exposure, the test solutions were continuously stirred by magnetic stirrers. - Initial cells density: 10000 cells/mL - No. of vessels per concentration (replicates): 3 - No. of vessels per control (replicates): 6- The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter®, Model ZM). TEST MEDIUM / WATER PARAMETERS - Source/preparation of dilution water: Reconstituted test water (OECD medium) according to OECD Guideline 201 OTHER TEST CONDITIONS - The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. - Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7370 Lux (range: 6170 to 8390 Lux, measured at nine places in the experimental area). EFFECT PARAMETERS MEASURED - Determination of cell concentrations: Algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.- At the end of the test, a sample was taken from the control and from the test concentrations of nominal 0.23, 0.11 and 0.050 mg/L to determine a potential influence of the test item on the shape and size of the algal cells by visual inspection. The second highest test concentration of 0.23 mg/L was chosen as highest test item concentration for inspection since the algal cell density at the nominal concentration of 0.50 mg/L was too low for a reliable examination.- Water Quality Criteria: The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily. TEST CONCENTRATIONS - Range finding study: The selection of the test concentrations was based on the results of range-finding tests (non-GLP). - Test concentrations: Nominal concentrations of the test item of 0.023, 0.050, 0.11, 0.23 and 0.50 mg/L (corresponding to 0.0069, 0.015, 0.033, 0.069 and 0.15 mg hydroxyethyl oleyl dimonium chloride/L corrected for water content)
Reference substance (positive control):
yes
Remarks:
potassium dichromate (Harlan Laboratories Study D25712)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.078 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.047 - 0.098 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.071 - 0.12 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.15 - 0.19 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.045 - 0.057 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.062 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.057 - 0.068 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.091 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.083 - 0.10 mg/L
Remarks:
Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.068 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Based on Chimexane CL
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.014 - 0.029 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.03 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.021 - 0.036 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.045 - 0.057 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.015 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.014 - 0.017 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.019 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.017 - 0.020 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.027 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CI: 0.025 - 0.030 mg/L
Remarks:
Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.014 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Based on hydroxyethyl oleyl dimonium chloride
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.02 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Based on hydroxyethyl oleyl dimonium chloride
Details on results:
- After 72 hours of test duration, the test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the mean measured concentration of 0.068 mg/L, corresponding to 0.020 mg hydroxyethyl oleyl dimonium chloride/L (results of Welch t-test and Williams t-test, respectively; one-sided smaller, α = 0.05). Thus, the 72-hour NOEC was determined to be the mean measured test item concentration of 0.045 mg/L, corresponding to 0.014 mg hydroxyethyl oleyl dimonium chloride/L, since up to and including this test concentration the growth of the algae was not statistically significantly inhibited compared to the control.- The microscopic examination of the algal cells at the end of the test revealed that the algal cells at the test concentrations of nominal 0.23 and 0.11 mg/L were affected (round-shaped instead of crescent-shaped). No differences in size and shape were determined for the algae growing at the next lower nominal test concentration of 0.050 mg/L and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to this concentration. - In the control, the biomass increased by a factor of 101 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control during 72 hours was 28%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.3%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled. - No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. - At the start of the test, the pH measured in the treatments was between 8.2 and 8.4. At the end of the test, pH values of 8.5 to 9.8 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test. The water temperature during the test was maintained at 22 °C.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in April 2011 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 1.3 mg/L (Harlan Laboratories Study D25712), range of the 72-hour EC50 for the growth rate from 2000 to 2011: 0.71-1.7 mg/L).
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression.For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test or Welch t-test.

Analysis of the Test Item Concentrations 

At the start of the test, the measured concentrations of Chimexane CL in the test media of the three highest nominal test concentrations of 0.11, 0.23 and 0.50 mg/L amounted to 87, 96, and 91% of the nominal values, respectively. Thus, the correct dosage of the test item could be verified. During the test period of 72 hours, a decrease of test item concentration in the test medium occurred. At the end of the test, the measured concentrations were below LOQ (limit of quantification, LOQ: 0.0416 mg/L) at the nominal test concentrations of 0.11 and 0.23 mg/L, and had decreased to 15% of the nominal value at the nominal concentration of 0.50 mg/L.

Thus, the biological results were related to the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and end of the test):

Nominal test concentration (mg/L)

Mean measured concentration of Chimexane CL (mg/L)

Mean measured concentration of hydroxyethyl oleyl dimonium chloride (mg/L)

0.023

NA

-

0.050

NA

-

0.11*

0.045

0.014

0.23*

0.068

0.020

0.50

0.182

0.055

n.a.: not analyzed; *: since the measured concentrations at the end of the test were <LOQ, the half LOQ was used as end value to calculate the geometric mean

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions and based on the mean measured concentrations of the test item, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.078, 0.10, 0.17, 0.045 and 0.068 mg/L, respectively (based on growth rate); 0.051, 0.062, 0.091, 0.045 and 0.068 mg/L, respectively (based on yield) [based on Chimexane CL].Under the test conditions and based on the mean measured concentrations of the test item, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.023, 0.030, 0.051, 0.014 and 0.020 mg/L, respectively (based on growth rate); 0.015, 0.019, 0.027, 0.014 and 0.020 mg/L, respectively (based on yield) [based on hydroxyethyl oleyl dimonium chloride].
Executive summary:

In an algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to the nominal concentrations of the test item of 0.023, 0.050, 0.11, 0.23 and 0.50 mg/L (corresponding to 0.0069, 0.015, 0.033, 0.069 and 0.15 mg hydroxyethyl oleyl dimonium chloride/L corrected for water content) were tested. Additionally, a control group was tested in parallel.

At the start of the test, the measured concentrations of Chimexane CL in the test media of the three highest nominal test concentrations of 0.11, 0.23 and 0.50 mg/L amounted to 87, 96, and 91% of the nominal values, respectively. Thus, the correct dosage of the test item could be verified. During the test period of 72 hours, a decrease of test item concentration in the test medium occurred. At the end of the test, the measured concentrations were below LOQ (limit of quantification, LOQ: 0.0416 mg/L) at the nominal test concentrations of 0.11 and 0.23 mg/L, and had decreased to 15% of the nominal value at the nominal concentration of 0.50 mg/L.

After 72 hours of test duration, the test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the mean measured concentration of 0.068 mg/L, corresponding to 0.020 mg hydroxyethyl oleyl dimonium chloride/L. Thus, the 72-hour NOEC was determined to be the mean measured test item concentration of 0.045 mg/L, corresponding to 0.014 mg hydroxyethyl oleyl dimonium chloride/L, since up to and including this test concentration the growth of the algae was not statistically significantly inhibited compared to the control.

Under the test conditions and based on the mean measured concentrations of the test item, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.078, 0.10, 0.17, 0.045 and 0.068 mg/L, respectively (based on growth rate); 0.051, 0.062, 0.091, 0.045 and 0.068 mg/L, respectively (based on yield) [based on Chimexane CL].

Under the test conditions and based on the mean measured concentrations of the test item, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.023, 0.030, 0.051, 0.014 and 0.020 mg/L, respectively (based on growth rate); 0.015, 0.019, 0.027, 0.014 and 0.020 mg/L, respectively (based on yield) [based on hydroxyethyl oleyl dimonium chloride].

Description of key information

Under the test conditions and based on the mean measured concentrations of the test item Chimexane CL, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.078, 0.10, 0.17, 0.045 and 0.068 mg/L, respectively (based on growth rate); 0.051, 0.062, 0.091, 0.045 and 0.068 mg/L, respectively (based on yield).

Under the test conditions and based on the mean measured concentrations of the test item, the EC10, EC20, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 0.023, 0.030, 0.051, 0.014 and 0.020 mg/L, respectively (based on growth rate); 0.015, 0.019, 0.027, 0.014 and 0.020 mg/L, respectively (based on yield) [based on hydroxyethyl oleyl dimonium chloride].

Key value for chemical safety assessment

EC50 for freshwater algae:
0.051 mg/L
EC10 or NOEC for freshwater algae:
0.023 mg/L

Additional information

A reliable algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP was available. The freshwater green algae speciesPseudokirchneriella subcapitata was exposed to the nominal concentrations of the test item of 0.023, 0.050, 0.11, 0.23 and 0.50 mg/L (corresponding to 0.0069, 0.015, 0.033, 0.069 and 0.15 mg hydroxyethyl oleyl dimonium chloride/L corrected for water content) were tested. Additionally, a control group was tested in parallel.

After 72 hours of test duration, the test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) at the mean measured concentration of 0.068 mg/L, corresponding to 0.020 mg hydroxyethyl oleyl dimonium chloride/L. Thus, the 72-hour NOEC was determined to be the mean measured test item concentration of 0.045 mg/L, corresponding to 0.014 mg hydroxyethyl oleyl dimonium chloride/L, since up to and including this test concentration the growth of the algae was not statistically significantly inhibited compared to the control.

The study was considered valid for that endpoint.