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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 02 October, 2012 to 23 November, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
: The NaHCO3 concentration of the test medium was 150 mg/L instead of 50 mg/L, as recommended by the OECD/EEC Guidelines, in order to maintain a more constant pH during the test and biomass (dw/mL) was not determined at the end of the test in the controls
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC, 1992. Algal growth inhibition test. Off. J. Of the European communities, L383 A/179, 19921229
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Sampling
At the start of the test, samples were taken from all test concentrations and the control and stock solution. The same procedure was followed at the end of the test.
At least 10 mL was sampled in each case and was diluted 1:1 with leaching solution. The samples were then gently shaken and transferred into the analytical vials for analysis. With each transfer step the pipettes used were rinsed with the solution to be analysed and the solution discarded before sampling for the actual analysis.
At the end of the test extractions of the test vessels at 0.019 and 0.120 mg/L were performed to establish the amount of test substance adsorbed to the test vessels that have contained algae. First test vessels were emptied and rinsed with deionized water. Then 10 mL leaching solution was added and the vessels were gently shaken.
Total extraction of the test solution (+ algae) and glassware was performed at the end of the study on test vessels containing the nominal test concentrations of 0.019 and 0.120 mg/L. For this purpose the whole sample volume of the two test concentrations was extracted by adding 40 mL leaching solution to the test medium in the Erlenmeyer. These vessels were then gently shaken.
Samples from the actual test replicates at the end of the test were filtered using a 0.45 µm filter to remove algae. Filters were primed with the relevant solution before use. All samples were diluted with leaching solution.


Chemical analysis
Chemical analyses were performed according to the analytical procedure described in annex 3. The samples were analyzed immediately after sampling.

Vehicle:
no
Details on test solutions:
Preparation of the stock solution
A first stock solution of 0.111 g/L of test material was prepared in a 100 mL volumetric flask. An accurately measured amount of 0.0111 g of test material, weighed out on an analytical balance, was loaded in 100 mL test medium. The stock solution was then ultrasonicated for 1 minute and a clear homogenous suspension was obtained. From this first stock a second stock solution of 1 mg/L was made. 0.90 mL was pipetted in 99.1 mL test medium.
The pH of the first stock solution was checked and found to be 8.3, and was not adjusted. This was not according to the study plan, but the deviation from the pH of the blank test medium was only 0.2 units and this deviation is therefore considered as minor.
The second stock solution, which was also a clear homogenous suspension, was used to prepare the test solutions.
The stock solution was stirred while adequate amounts were taken for test solutions.

Preparation of the test solutions
The test solutions were prepared by addition of the adequate amounts of the second stock solution to the test vessels, then dilution water was added up to the required final volume.
Based on the results of a range finding study which gave no inhibition at 0.01 mg/L and 100% inhibition at 0.1 mg/L the following final nominal test concentrations were prepared: 0.005, 0.011, 0.022, 0.046 and 0.097 mg/L. Because 50% effect on growth rate was not reached it was decided to repeat the test with a different concentration range: 0.008, 0.019, 0.048, 0.120 and 0.300 mg/L.
Control vessels containing only test medium were included in the test.
All test vessels were closed with glass stoppers.



Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata (freshwater unicellular algae (CCAP 278/4)
- Source (laboratory, culture collection): Culture collection of algae and protozoa SAMS Research Services Ltd. Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland
- Method of cultivation: Cultures on sloped agar tubes were stored at 4°C in the dark until required. Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
The de-ionised water used in the study contained less than 10 µg/L of copper (not measured under GLP)
Test temperature:
23 ± 2°C
pH:
8.3 to 11.1
Dissolved oxygen:
Less than 2.0 mg/L NPOC-content
Salinity:
Standard OECD medium
Conductivity:
Less than 5 μS/cm
Nominal and measured concentrations:
Nominal: 0.008, 0.019, 0.048, 0.120 and 0.300 mg/L
Measured initial: 0.005, 0.011, 0.031, 0.080, and 0.205 mg/L
Geometric mean: 0.005*, 0.008, 0.019, 0.039 and 0.065 mg/L
* Based on C16 measurements only.
Details on test conditions:
-Test flasks
The test was performed in 100 mL Erlenmeyer flasks containing 40 mL of medium. The test flasks were closed with glass stoppers.
-General test principles and procedures   
Culture medium was prepared by diluting the stock mineral salts in an appropriate vessel. This medium was sterilized by filter sterilization (0.2 µm). Adequate amounts of test substance were added and allowed to disperse (See section 4.3 for more details about the preparation of the test solutions). The Erlenmeyer flasks were then filled with test solution up to a total volume of 40 mL using a 50 mL syringe. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control were included.
The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer

- Apparatus
The pH was determined with a microcomputer pH meter. The temperature was measured with a continually measuring thermocouple. The light intensity was measured with a light intensity meter. The purity of the algae was determined with a microscope.

- Culturing cabinet and test conditions
The test was carried out in a temperature-controlled illuminated orbital incubator, in which the temperature was maintained at 23 ± 2°C, and a continuous uniform illumination was provided in the spectral range of 400 to 700 nm by using fluorescent lamps at a distance of about 0.36 +/-0.02 m from the algal cultures. The light intensity was in the range of 60 to 120 μE·m-2·s-1 (= μmol.m-2·s-1). The test vessels were swirled continuously at a speed sufficient to prevent sedimentation of the algae.

- Determination of cell concentrations
Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction of light and cell density, a calibration curve was made which is checked yearly. From the relation between extinction (E) and counted cell number per mL (N) the following calibration curve was determined using linear regression:
 N = 2.000×106´E – 1.81×105  (R2= 0.993)
The calibration curve was used to determine the cell density of the inoculum before and during the test when needed.
 
- Determination of pH, temperature, light, and purity of algae
The pH of all samples and controls were measured at the beginning (t=0 h) and at the end (t=72 h) of the test. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. The light intensity was measured at the beginning and at the end of the test. At the end of the test five random samples were microscopically checked for purity of the algal culture.

- Quality control of the algae
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when the cultures are grafted from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, and found to be between the set EC50 values of 0.25 to 2.0 mg/L.

- Evaluation of data
The mean values of the extinction of light for each test substance concentration were used to calculate the growth and specific growth rate. Details of the calculations are given in Annex 6 (refer to the PDF under the 'attached background material').
Where possible, the EC10,20,50,80 values were computed from the best fitted line (leastsquares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test (Ref 4) The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller’s theorem if possible (Ref 5). All computations were
performed using TOXCALCTM version 5.023 program.

- Quality criteria:
The following quality criteria have been met in the present study:
􀁸 The cell density of the controls increased at least a factor 16 within 72 h
􀁸 The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.
􀁸 The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.
􀁸 The EC50 values of the reference compound, potassium dichromate, should be in the range of 0.25-2.0 mg/L.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate 99.5% purity lot H0234 JT baker
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.094 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits of 0.020 to 0.129 mg/L
Remarks:
(i.e., equivalent to 0.068 mg a.i./L)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.155 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits of 0.091 to 0.185 mg/L
Remarks:
(i.e., equivalent to 0.113 mg a.i./L)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.115 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits of 0.098 and 0.152 mg/L.
Remarks:
(i.e., equivalent to 0.084 mg a.i./L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (i.e.,equivalent to 0.058 mg a.i./L)
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.205 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
- Toxicity:
The results of the test are presented in Annex 3. The graphical presentations of the test results are given in Figures 1 and 2 of that Annex.
The results reported are based on measured initial test substance concentrations. All reported concentrations refer to the test substance as received and are thus not corrected for active ingredient content. The test guideline (Ref 1) used for this test states that for adsorbing substances the
endpoints could be based on measured initial concentration when can be shown that decrease in test concentration of the test substance in the course of the test is not accompanied by a decrease in growth inhibition. The test substance tested here is a surface active substance which adsorbs strongly onto negatively charged surfaces as glassware and algae.
In the Figure below is shown that a decrease in growth inhibition does not occur due to a decrease in test concentration during the test The average growth rate per test concentration did not increase in time, meaning that the inhibition did not decrease, as the growth rate for 48 to 72h is lower than for 24 to 48h. As for almost all test concentration no recovery of growth was observed in time, it is justified to base the dose-response relation on measured initial concentrations as these are causing the effects observed.
No significant effect was observed on growth rate up to 0.08 mg/L. The NOEC is therefore 0.08 mg/L and LOEC is 0.205 mg/L. The ErC10 is 0.094 mg/L, with 95% confidence limits of 0.020 to 0.129 mg/L. The ErC50 is 0.155 mg/L, with 95% confidence limits of 0.091 to 0.185 mg/L. The EbC50 was found to be 0.115 mg/L, with 95% confidence limits of 0.098 and 0.152 mg/L. The statistics and growth-response curves are presented in Annex 4. The critical endpoints and parameters are summarized in Table IV of Annex 3.


- pH, temperature, light and purity of algae:
The pH measurements showed a maximum increase of 3.4 pH units. This is more than recommended in the guideline, but was caused by the closed test vessels. As all validity criteria for the control were met, growth was within the required criteria. The pH increase is therefore considered to have no influence on the integrity of the test results.
The temperature varied from 21.8 to 22.3 °C during the test.
Light intensity was 102.8 µmol.m-2.s-1 both at the beginning and end of the test. Both are in accordance with the required conditions described in the study plan.
The five random samples were all pure and not contaminated with bacteria

- Chemical analysis
The concentration of the test substance present in the test vessels were quantified using the method as described in Annex 5 (presented in the attached PDF). The concentration of test substance is recalculated, correcting for the difference of C16 and C18 present in the test substance. See the first Table in the attached PDFfor a summary of the measured test concentrations.
The stock solutions used to prepare the test solutions showed a good recovery of >=94%. The recovery of the test concentrations were as expected all below 80% of the nominal concentrations. The result of the sample from the lowest test concentration taken at the end of the test was below LOQ for C18. The measured concentration given is therefore only based on the measured concentration of C16. Measured initial concentrations are used for endpoint calculations (due to the reasons explained under Toxicity). The analytical method met all quality criteria set (see second Table in the attached PDF).

- Validity criteria:
The validity of the test was shown by:
• the increase of the extinction of the control over 72 h by a factor of 61
• the mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%.
• the coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures did not exceed 7%.

Refer to the attached PDF for details on results.



No significant effect was observed on growth rate up to 0.08 mg/L. The NOEC is therefore 0.08 mg/L and LOEC is 0.205 mg a.i./L. The ErC10 is 0.094 mg/L, with 95% confidence limits of 0.020 to 0.129 mg a.i./L. The ErC50 is 0.155 mg a.i./L, with 95% confidence limits of 0.091 to 0.185 mg/L. The EbC50 was found to be 0.115 mg a.i./L, with 95% confidence limits of 0.098 and 0.152 mg/L.





Refer annexure 2 for details (in the attached backgroud material).

Results with reference substance (positive control):
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when the cultures are grafted from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, and found to be between the set EC50 values of 0.25 to 2.0 mg/L
Reported statistics and error estimates:
Where possible, the EC10,20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test.The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller’s theorem if possible (Ref 5). All computations were performed using TOXCALCTM version 5.023 program.

Deviations from the study plan

There are two deviations from the study plan:

1. In the second test the test vessels were closed with glass stoppers to facilitate the extraction at the end of the test.

2.The pH of the first stock solution was checked and found to be 8.3, and was not adjusted. This was not according to the study plan, but the deviation from the pH of the blank test medium was only 0.2 units and this deviation is therefore considered as minor.

Validity criteria fulfilled:
yes
Conclusions:
ased on the results of the read across study the 72 h ErC10 and ErC50 values of the test substance based on effects on growth rate of aquatic algae is considered to be 0.094 (i.e., 0.068 mg a.i./L) and 0.155 mg/L (i.e., 0.113 mg a.i./L) (measured initial), respectively
Executive summary:

A study was conducted to evaluate the toxicity of the read-across substance, C16-18 TMAC (72.78% active), to Pseudokirchnerella subcapitata according to OECD Guideline 201, in compliance with GLP. The toxicity of the read across substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours under static conditions. Algae were exposed to the read across substance at nominal concentrations of 0.008, 0.019, 0.048, 0.120 and 0.300 mg/L. Algal cell concentrations were determined spectrophotometrically. Analytical verification of the test concentrations was performed using LC-MS/MS at the beginning and end of the test. The results of the chemical analyses showed that the concentrations in the test solutions at the start and at the end of the test in the test vessels were all below 80% of the nominal, which is expected because surfactant-based substances are known to bind to algae cells. However, as growth rates per test concentration did not decrease in time compared to the control, the measured initial concentrations were used to calculate the effects, in line with the guideline recommendations. The measured initial concentrations were determined to be 0.005, 0.011, 0.031, 0.080, and 0.205 mg/L (i.e., equivalent to 0.0037, 0.008, 0.023, 0.058 and 0.149 mg a.i./L respectively). No significant effect was found on growth rate up to 0.08 mg/L, therefore the NOEC was at 0.08 mg/L and LOEC was at 0.205 mg/L. The ErC10 and ErC50 for growth rate was determined to be at 0.094 mg/L (with 95% confidence limits of 0.020 to 0.129 mg/L) and 0.155 mg/L (with 95% confidence limits of 0.091 to 0.185 mg/L). The EbC10 and EbC50 for biomass was determined to be at 0.036 mg/L (with 95% confidence limits of 0.031 to 0.040 mg/L) and 0.047 mg/L (with 95% confidence limits of 0.043 to 0.055 mg/L). All validity criteria were fulfilled. Under the study conditions, the 72 h ErC10 and ErC50 values of the read across substance based on effects on growth rate of aquatic algae were determined to be 0.094 (i.e., 0.068 mg a.i./L) and 0.155 mg/L (i.e., 0.113 mg a.i./L) (measured initial), respectively (Gyimesi M et al., 2012). Based on the results of the read across study, similar effect levels are considered for the test substance.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
no
Analytical monitoring:
yes
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23.7 to 24.6°C
pH:
8.2 to 9.1
Nominal and measured concentrations:
Nominal concentrations: 0.035, 0.071, 0.142, 0.283 and 0.566 mg/L
Measured (concentrations were only measured for the low, middle and high concentrations):
0.03, 0.14 and 0.51 mg/L at 0 h, respectively and
0.01, 0.03 and 38 mg/L at 96 h, respectively.
Details on test conditions:
Based on the results of a range-finding test, Selenastrum capricornutum was exposed to the test substance at nominal concentrations of 0.035, 0.071, 0.142, 0.283 and 0.566 mg/L. The toxicity of the test substance to exponentially growing Selenastrum capricornutum was determined over an exposure period of 96 h. The test was conducted in a mineral salts medium at temperatures ranging from 23.7 to 24.6ºC in an illuminate orbital incubator. The pH in the test media varied from 8.2 to 9.1. The test was performed using six replicates for the control and three replicates for each concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.113 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: equivalent to 0.099 mg a.i./L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.177 mg/L
Nominal / measured:
nominal
Basis for effect:
growth rate
Remarks on result:
other: equivalent to 0.156 mg a.i./L
Details on results:
96-h EbC50 = 0.11 mg/L (0.11 - 0.12 mg/L)
96-h ErC50 = 0.18 mg/L (0.17 - 0.19 mg/L)
Results with reference substance (positive control):
EbC50 and ErC50 values of the reference compound, potassium dichromate were 0.8 and 1.5 mg/L, respectively

The test was valid as shown by the EbC50 and ErC50 values of the reference compound, potassium dichromate (0.8 and 1.5 mg/L, respectively), the increase of the extinction of the control over 72 h by a factor of 16 and by a maximum deviation of the pH of 0.9 units. Chemical analysis, using HPLC, of duplicate samples taken at the beginning of the test indicated that the exposure concentrations were substantially achieved (85 - 99% of the nominal values). The concentrations at the end of the test were strongly decreased (21 -67% of the nominal values), probably due to adsorption of the test substance to the test flask walls. Thus, EC values were calculated based on the nominal, rather than measured, concentrations.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 96 h EbC50 and ErC50 values for effects on growth and growth rate were determined to be 0.113 and 0.177 mg/L (i.e., equivalent to 0.099 and 0.156 mg a.i./L) respectively. The NOEC and LOEC values for both parameters were identified to be at 0.035 and 0.071 mg/L (i.e., equivalent to 0.03 and 0.062 mg a.i./L) respectively.
Executive summary:

A study was conducted to evaluate the toxicity of the read across substance, tricetylmethyl ammonium chloride (87.9% active), to Selenastrum capricornutumaccording under static conditions, according to OECD Guideline 201, EU Method C.3 and ISO 8692, in compliance with GLP. Based on the results of a range-finding test,Selenastrum capricornutumwas exposed to the read across substance at nominal concentrations of 0.035, 0.071, 0.142, 0.283 and 0.566 mg/L (i.e., 0.03, 0.62, 0.125, 0.245 and 0.498 mg a.i./L). Chemical analysis, using HPLC, of duplicate samples was taken at the beginning of the test indicated that the exposure concentrations were substantially achieved (85-99% of the nominal values). The concentrations at the end of the test were strongly decreased (21 -67% of the nominal values), probably due to adsorption of the read across substance to the test flask walls. Thus, EC values were calculated based on the nominal, rather than measured, concentrations. The toxicity of the read across substance to exponentially growing Selenastrum capricornutum was determined over an exposure period of 96 h. The test was performed using six replicates for the control and three replicates for each concentration. All the validity criteria were fulfilled. Under the conditions of the study, the 96 h EbC50 and ErC50 values for effects on growth and growth rate were determined to be 0.113 and 0.177 mg/L (i.e., equivalent to 0.099 and 0.156 mg a.i./L) respectively. The NOEC and LOEC values for both parameters were identified to be at 0.035 and 0.071 mg/L (i.e., equivalent to 0.03 and 0.062 mg a.i./L) respectively. (TRS (HPV), 2001). Based on the results of the read across study, similar effect levels can be expected for the test substance.

Description of key information

Based on the results of the read across studies, the 72-h ErC50 and ErC10 values, which was determined at 0.113 and 0.068 mg a.i./L, for effects on growth rate of algae have been considered further for hazard/risk assessment of the test substance. 

Key value for chemical safety assessment

EC50 for freshwater algae:
113 µg/L
EC10 or NOEC for freshwater algae:
68 µg/L

Additional information

Study 1: A study was conducted to evaluate the toxicity of the read-across substance, C16-18 TMAC (72.78% active), to Pseudokirchnerella subcapitata according to OECD Guideline 201, in compliance with GLP. The toxicity of the read across substance to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours under static conditions. Algae were exposed to the read across substance at nominal concentrations of 0.008, 0.019, 0.048, 0.120 and 0.300 mg/L. Algal cell concentrations were determined spectrophotometrically. Analytical verification of the test concentrations was performed using LC-MS/MS at the beginning and end of the test. The results of the chemical analyses showed that the concentrations in the test solutions at the start and at the end of the test in the test vessels were all below 80% of the nominal, which is expected because surfactant-based substances are known to bind to algae cells. However, as growth rates per test concentration did not decrease in time compared to the control, the measured initial concentrations were used to calculate the effects, in line with the guideline recommendations. The measured initial concentrations were determined to be 0.005, 0.011, 0.031, 0.080, and 0.205 mg/L (i.e., equivalent to 0.0037, 0.008, 0.023, 0.058 and 0.149 mg a.i./L respectively). No significant effect was found on growth rate up to 0.08 mg/L, therefore the NOEC was at 0.08 mg/L and LOEC was at 0.205 mg/L. The ErC10 and ErC50 for growth rate was determined to be at 0.094 mg/L (with 95% confidence limits of 0.020 to 0.129 mg/L) and 0.155 mg/L (with 95% confidence limits of 0.091 to 0.185 mg/L). The EbC10 and EbC50 for biomass was determined to be at 0.036 mg/L (with 95% confidence limits of 0.031 to 0.040 mg/L) and 0.047 mg/L (with 95% confidence limits of 0.043 to 0.055 mg/L). All validity criteria were fulfilled. Under the study conditions, the 72 h ErC10 and ErC50 values of the read across substance based on effects on growth rate of aquatic algae were determined to be 0.094 (i.e., 0.068 mg a.i./L) and 0.155 mg/L (i.e., 0.113 mg a.i./L) (measured initial), respectively (Gyimesi M et al., 2012).

Study 2: A study was conducted to evaluate the toxicity of the read across substance, tricetylmethyl ammonium chloride (87.9% active), to Selenastrum capricornutumaccording under static conditions, according to OECD Guideline 201, EU Method C.3 and ISO 8692, in compliance with GLP. Based on the results of a range-finding test,Selenastrum capricornutumwas exposed to the read across substance at nominal concentrations of 0.035, 0.071, 0.142, 0.283 and 0.566 mg/L (i.e., 0.03, 0.62, 0.125, 0.245 and 0.498 mg a.i./L). Chemical analysis, using HPLC, of duplicate samples was taken at the beginning of the test indicated that the exposure concentrations were substantially achieved (85-99% of the nominal values). The concentrations at the end of the test were strongly decreased (21 -67% of the nominal values), probably due to adsorption of the read across substance to the test flask walls. Thus, EC values were calculated based on the nominal, rather than measured, concentrations. The toxicity of the read across substance to exponentially growing Selenastrum capricornutum was determined over an exposure period of 96 h. The test was performed using six replicates for the control and three replicates for each concentration. All the validity criteria were fulfilled. Under the conditions of the study, the 96 h EbC50 and ErC50 values for effects on growth and growth rate were determined to be 0.113 and 0.177 mg/L (i.e., equivalent to 0.099 and 0.156 mg a.i./L) respectively. The NOEC and LOEC values for both parameters were identified to be at 0.035 and 0.071 mg/L (i.e., equivalent to 0.03 and 0.062 mg a.i./L) respectively (Kroon and Geurts (1994) study cited in TRS (HPV), 2001).

Based on the results of the read across studies, the lower and more recent 72-h ErC50 and ErC10 values, which was determined at 0.113 and 0.068 mg a.i./L, for effects on growth rate of algae have been considered further for hazard/risk assessment of the test substance.