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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 10, 1996 to June 21, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
analysis in test vessels was not conducted due to loss of substance causing the concentration to fall below detection limit. This does mean the actual exposure concentration changed with time due to adhesion of the substance to algae.
GLP compliance:
yes
Specific details on test material used for the study:
- Sample code: T96005
- Batch/lot No:016950793 1900
- Purity: 50.2%
- Composition: light yellow liquid
- Water solubility:soluble in water
- Vapour pressure:12.1 kPa (50°C)
Analytical monitoring:
yes
Details on test solutions:
A test substance stock suspension was prepared by weighing out the test substance using a analytical balance [AE 163, Mettler-Toledo AC, Greifensee, Switzerland Standard Operation Procedure K4 (4.13). For the definitive test 0.1009g test substance was mixed in a limited amount of mineral salts medium and mixed for 5 min. The pH was adjusted to pH=7.3 using 1 M sodium hydroxide. Finally, the solution was filled up to 100 mL in a volumetric flask with mineral salts medium.

For the definitive test an amount of culture medium was prepared by diluting the stock mineral salts in a 3-L vessel. This medium was sterilized by filter sterilization (0.2 pm). The inoculum was added from an exponentially growing culture. Various amounts of of the stock solution of the test substance were added to the test vessels (100 mL Erlenmeyers) in order to obtain the following nominal concentrations (triplicate): 0.005, 0.015, 0.045, 0.136 and 0.407 mg/L. The test vessels were filled with inoculated medium upto a total volume of 40 mL using a sterilized dispenser. In addition, six replicate of the control were included. The extinction in each Erlenmeyer was measured after 0, 24, 48, 72 and 96h. Algal medium was used as a blank in the spectrophotometer.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Preparation of the inoculum:
The initial stock culture was inoculated with Selenastrum capricornutum from a sloped agar tube and checked for purity by microscopic means.
This algal stock culture (40 mL) of Senostrum capricornutum was regularly transferred to fresh mineral medium to act as inoculum for the test.

The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described above From this algal culture a dilution was prepared to obtain an initial cell denslty of 10,000 cells/mL in the medium of the definitive test.

The quality of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) simultaneously with the definitive test.
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Post exposure observation period:
Five random samples were microscopically examined for bacterial contamination after the test. No contamination was observed.
Test temperature:
21.6 to 23.0°C
pH:
8.5 to 10.0
Salinity:
The deionized water used to make algae medium had a conductivity of less than 5 P S.cm and a TOC content of less than 2 mg/L. This water was produced from tap water in a water purification system (Spectrum-Elgastat, Breukelen, The Netherlands). For full media composition, refer to the report.
Nominal and measured concentrations:
0.005, 0.015, 0.045, 0.136 and 0.407 mg/L (nominal)
A 1.0 mg/L-concentration was measured before and after test to determine stability over test period (no algae).
Details on test conditions:
The culturing apparatus was a temperaturecontrolled illuminated orbital incubator (Gallenkamp INR 401, Breda, The Netherlands). Standard Operation Procedure K 1 1 (4.8) in which the temperature was maintained at ca. 25°C and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using 30 W fluorescent lamps of the type ‘universal white’ (colour temperature of approximately 4000 K), at a distance of 0.35 m from the algal cultures. The culture flasks were rotated continuously at 100 rev/min to prevent sedimentation of the algae.
The test was performed in 100 mL Erlenmeyers containing 40 mL of mineral salts medium. The test flasks were closed with cotton-wool stoppers.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.01 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 0.02 - 0.03; 95% confidence limits
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.03 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.06 - 0.07; 95% confidence limits
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
0.011 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
0.002 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
- Exponential growth in control: Yes
- Flocculation: No
- Adherence to test vessels: No
- Any stimulation of growth found in any treatment: No
Results with reference substance (positive control):
The definitive test is valid as shown by the EbC50 and Erc50 values of the reference compound, potassium dichromate (0.95 and 1.8 mg/L, respectively), the increase of the extinction of the control over 72 h by a factor of 64 and by a maximum deviation of the pH of 1.5 unit.
Reported statistics and error estimates:
All computations were performed using the Akzo programme ‘Algal’ (version 3.3) made in SAS (4.7).

Table 1. Summary of results absorbance

 Nominal concentration mg/L (mean)  0h  96h
 Control  0.006  1.031
 0.005  0.006  0.971
 0.015  0.006  0.631
 0.045  0.007  0.308
 0.136  0.007  0.020
 0.407  0.006  0.009
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the EbC50, and ErC50 (0-96h) of the test substance were 0.02 mg/L (i.e., approximately equivalent to 0.01 mg a.i./L) and 0.06 mg/L (i.e., approximately equivalent to 0.03 mg a.i./L), respectively. The LOEC determined from the results for the test substance was 0.0025 mg a.i./L and the NOEC derived from the results for test substance was <0.0025 mg a.i./L.
Executive summary:

A study was conducted to determine the toxicity to aquatic algae of the test substance (50.2% purity) according to OECD Guideline 201, in compliance with GLP. Selenastrum capricornutum were exposed for 96 h to nominal concentrations of 0, 0.005, 0.015, 0.045, 0.136 and 0.407 mg/L of test substance. Analytical dose verification was performed. Chemical analysis of duplicate samples taken at the beginning (96% recovery) and at the end (98% recovery) of the test indicated that the exposure concentrations were substantially achieved. Growth rate and biomass were recorded throughout the experiment. Under the study conditions, the EbC50 and ErC50 (0-96 h) were 0.02 mg/L (equivalent to 0.01 mg a.i./L) and 0.06 mg/L (equivalent to 0.03 mg a.i./L), respectively. The LOEC was 0.0025 mg a.i./L and the NOEC was < 0.0025 mg a.i./L (Kroon, 1996).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): Alkyldimethylbenzylammonium chloride (ADBAC); (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: slightly yellowish liquid
- Analytical purity: 49-51% (average 50%)
- Impurities (identity and concentrations): Alkyldimethylbenzylammonium chloride in water
- Storage condition of test material: Room temperature
- Radiochemical purity (if radiolabelling): 99.7%
- Specific activity (if radiolabelling): 25 mCi/mmol
- Locations of the label (if radiolabelling): [14C]-alkyldimethylbenzylammonium chloride
Analytical monitoring:
yes
Details on sampling:
Samples from the controls and the test flasks containing the different test substance concentrations were taken after 0, 23, 47 and 71h. 5 mL samples of cultures were added to 10 mL of Ultima Gold XR scintillation liquid.
Vehicle:
yes
Details on test solutions:
- For the range-finding test, an amount of 20.9 mg test substance was dissolved in 100 mL of algal medium. Dilutions were then prepared in medium so as to yield a test substance concentrations series of 0, 10, 104, 1,040 and 10,400 µg/L.
- For the growth inhibition test, an amount of 13.7 mg test substance was dissolved in 500 mL algal medium. Dilutions were the prepared in medium to yield a nominal test substance concentration series of 0, 3.7, 13, 24, 43, 137 and 435 µg/L.
- For the 14C-spiking stock solution, an amount of 10 µL of 14C-labelled test substance solution (containing 47760 Bq) was added to 10 mL ultrapure water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The fresh water green alga Selenastrum capricornutum (CCAP 278/4)
The culture was supplied by CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.

A pre-culture of algae in exponential growth phase was prepared as per the OECD guideline no. 201 using medium as described in the OECD guideline.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22 - 23 °C (average = 22.3°C)
pH:
8.2-8.3 (in the absence of algal cell)
8.2-8.6 (in the presence of algal cell)
Nominal and measured concentrations:
Nominal test concentrations: 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L.
Measured concentrations:
Details on test conditions:
An algal inoculum was added to each flask to achieve nominal density of 10,000 cells/mL. Nominal test concentrations were 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L. These concentrations refer to the total of labelled and non-labelled test substance and after addition of algal substance. Flasks were continuously shaken at 100 rpm. The test was carried out at 22.3°C average, and a pH of 8.2. Illumination was 60-120 µmol s-1.m-2 using fluorescent lights. Algal cell densities were determined in the treatments and control at 0, 23, 47 and 71h. The pH was measured in all cultures after 71h. All other parameters were also within the guideline recommendations.
Test was carried out in triplicate with six controls containing algae only and a single background series containing test substance without algae. Algal densities and biovolume were determined after 0, 23, 47 and 71h. The measured values were corrected for the background values in appropriate blanks. The mean values were used for further calculations.
The algal growth was determined by electronic particle counting after 0, 23, 47 and 71h incubation. The test fulfilled the validity criteria of sufficient growth and a minimum increase of test medium pH. The effect values were calculated using a statistic parametric model.
Reference substance (positive control):
yes
Remarks:
The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
49 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 40 - 61; 95% confidence limits
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 11 - 22; 95% confidence limits
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
<= 1.2 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: growth rate inhibition was only 3.1% at this level which was considered negligible
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic inspection of morphology of algal cells in pre-culture at the start of the test revealed normal cells. At the end of the test in the cultures treated with nominal test concentrations of 24 and 43 µg/L, small pale cells were observed. At higher test substance concentrations, no algae were found.
- Flocculation: No
- Adherence to test vessels: No
- Any stimulation of growth found in any treatment: No
Results with reference substance (positive control):
The test system used is checked with reference substances (K2Cr2O7 and/or 3,5-dicholorophenol) on an approximately yearly basis. The result of these reference tests demonstrate a constant quality of the test system during an extended period of at least 15 years. The resulting EC50 values are also comparable to the mean values found in international ring tests.
Reported statistics and error estimates:
Parametric model

- The range-finding test revealed that inhibiting effects could be expected at nominal test substance concentrations higher than 10 µg/L.

- The control growth rate (0.065/h) is higher than the minimum cell multiplication factor of 16 during a three d test given in the guideline (corresponding to a growth rate of 0.038/h).

- The highest pH increase in the test medium was 0.4, which is well below the limit given in the guideline.

- The test therefore met the validity criteria of the guideline.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditons, the 72h ErC50 and EbC50 (Selenastrum capricornutum) of the test substance were 49 µg a.i./L and 14 µg a.i./L, respectively. The 72h NOEC determined from the results for the test substance was equal to or less than 1.2 µg a.i./L (i.e., equivalent to 0.0012 mg a.i./L). Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only.
Executive summary:

A study was conducted to determine the toxicity to aquatic algae of the test substance according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Selenastrum capricornutum were exposed for 72 h at nominal concentrations of 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L. Concentrations were analytically determined at the start of the test and after 23, 47 and 72 h. Algal growth was measured by electronic particle counting after 0, 23, 47 and 72 h. Test substance concentrations were found to be relatively stable during the test and the mean measured test substance concentrations of 0, 1.2, 5.1, 11, 22, 98 and 382 µg/L were used for calculation of effect parameters. Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only. Under the study conditions, the 72h ErC50 and EbC50 were 49 and 14 µg a.i./L, respectively. The 72 h NOEC was ≤1.2 µg a.i./L (Mayer, 2001).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 05 March, 2004 to 19 March, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The aquatic alage study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Physical state: A clear, light yellow liquid with a faint/slight alcoholic odour
- Composition of test material, percentage of components: ADBAC C12-14 content of 79.75%,
- Date received: 03-03-2004
- Stability under test conditions: stable under test conditions
- Storage condition of test material: at 20°C
- Other:
- Sloubility: Soluble in water
- Specific gravity: 0.976 mg/L at 15°C
Analytical monitoring:
not specified
Test organisms (species):
Skeletonema costatum
Details on test organisms:
The strain NIVA BAC 1 was used.
Algal medium 1/2 'T' from Guillard and Ryther (1962) was used.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
20± 2.4°C
pH:
8.51 to 8.64
Nominal and measured concentrations:
0.1, 0.5, 2.5 and 10 mg/L (nominal whole product)

.
Details on test conditions:
Test method:
- Growth inhibition test (72h) with the marine diatom Skeletonema costatum was performed
- Initially, a preliminary (range finding) test with two parallel test concentrations, followed by a definitive test with 3 parallel concentrations and 6 controls was performed.
- Inhibition was measured as a reduction in growth rate, (the growth rate is the increase in cell density per unit time) relative to control cultures grown under identical conditions.
The EC50-, EC90- , EC10 - and NOEC- values were determined. The EC50-value is defined as the dosage of the test substance that gives a 50% reduction in growth rate relative to control. The NOEC-value is defined as the highest dosage of the test substance that gives no observed effect on the growth rate. The cell density was measured by fluorescence (Turner 10-100 R fluorometer).
The average specific growth rate (mav) is calculated based on this equation:
mav =In (Xt)- In (Xo)/t
Xt= final algal density
Xo= initial algal density
T= test
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
0.43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.04 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.032 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.096 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
In this test, all the control parameters were within the recommended ranges. According to the EEC classification system (EEC, 1993), a test substance is classified as very toxic if EC50 < 1 mg/L, as toxic if EC50 = 1-10 mg/L, and as harmful if EC50 >10-100 mglL based on algae. Based on this, the test substance PR-4951 can be classified as very toxic according to the EEC classification system
Results with reference substance (positive control):
One test concentration (1.5 mg/L) of the reference substance, 3,5-dichlorphenol, was analyzed. The growth rate inhibition observed was 40% after 72h.
Reported statistics and error estimates:
Method for data handling: The mean values of EC50, EC90, EC10, NOEC and the confidence limits are calculated from the computer program Excel. The dosage-response curve is printed out, and the EC-values are estimated by drawing lines by hand
Validity criteria fulfilled:
yes
Conclusions:
Based on the results of the read across study, the 72h ErC50 and ErC10 (Skeletonema costatum) of the test substance were 0.26 and 0.12 mg/L, respectively (i.e., equivalent to 0.207 mg a.i./L and 0.096 mg a.i./L). The 72h NOEC was 0.04 mg/L which corresponds to 0.032 mg a.i./L.
Executive summary:

A study was conducted to determine the toxicity to marine algae of the read across substance (79.75% purity), benzyl-C12-14-alkyldimethylammonium chloride (C12-14 ADBAC), according to ISO 10253, in compliance with GLP. Skeletonema costatum were exposed for 72 h at nominal concentrations of 0, 0.1, 0.5, 2.5 and 10 mg/L. Analytical dose verification was not performed. The cell density was measured by fluorescence. Under the study conditions, the 72 h ErC50 and ErC10 were 0.26 and 0.12 mg/L (equivalent to 0.207 and 0.096 mg a.i./L), respectively. The 72 h NOEC was 0.04 mg/L, corresponding to 0.032 mg a.i./L (Sundfor, 2004).

Description of key information

Based on effects on growth rate, the 72 h ErC50 and ErC10 values for fresh water algae, Pseudokirchneriella subcapitata, due to the test substance were determined to be 0.03 and 0.009 mg a.i./L (nominal) respectively. The corresponding values for marine wateralgae, Skeletonema costatum, were determined to be 0.207 and 0.096 mg a.i./L (nominal) respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.03 mg/L
EC50 for marine water algae:
0.207 mg/L
EC10 or NOEC for freshwater algae:
0.009 mg/L
EC10 or NOEC for marine water algae:
0.096 mg/L

Additional information

Study 1. A study was conducted to determine the toxicity to aquatic algae of the test substance (50.2% purity) according to OECD Guideline 201, in compliance with GLP. Selenastrum capricornutum were exposed for 96 h to nominal concentrations of 0, 0.005, 0.015, 0.045, 0.136 and 0.407 mg/L of test substance. Analytical dose verification was performed. Chemical analysis of duplicate samples taken at the beginning (96% recovery) and at the end (98% recovery) of the test indicated that the exposure concentrations were substantially achieved. Growth rate and biomass were recorded throughout the experiment. Under the study conditions, the EbC50 and ErC50 (0-96 h) were 0.02 mg/L (equivalent to 0.01 mg a.i./L) and 0.06 mg/L (equivalent to 0.03 mg a.i./L), respectively. The LOEC was 0.0025 mg a.i./L and the NOEC was < 0.0025 mg a.i./L (Kroon, 1996).

Study 2. A study was conducted to determine the toxicity to aquatic algae of the test substance according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Selenastrum capricornutum were exposed for 72 h at nominal concentrations of 0, 3.7, 13, 24, 43, 136 and 431 µg a.i./L. Concentrations were analytically determined at the start of the test and after 23, 47 and 72 h. Algal growth was measured by electronic particle counting after 0, 23, 47 and 72 h. Test substance concentrations were found to be relatively stable during the test and the mean measured test substance concentrations of 0, 1.2, 5.1, 11, 22, 98 and 382 µg/L were used for calculation of effect parameters. Statistical assessment of data by application of parametric model indicated a significant effect on the growth rate only. Under the study conditions, the 72h ErC50 and EbC50 were 49 and 14 µg a.i./L, respectively. The 72 h NOEC was ≤1.2 µg a.i./L (Mayer, 2001).

Study 3. A study was conducted to determine the toxicity to marine algae of the read across substance (79.75% purity), benzyl-C12-14-alkyldimethylammonium chloride (C12-14 ADBAC), according to ISO 10253, in compliance with GLP. Skeletonema costatum were exposed for 72 h at nominal concentrations of 0, 0.1, 0.5, 2.5 and 10 mg/L. Analytical dose verification was not performed. The cell density was measured by fluorescence. Under the study conditions, the 72 h ErC50 and ErC10 were 0.26 and 0.12 mg/L (equivalent to 0.207 and 0.096 mg a.i./L), respectively. The 72 h NOEC was 0.04 mg/L, corresponding to 0.032 mg a.i./L (Sundfor, 2004).