Registration Dossier
Registration Dossier
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EC number: 201-579-4 | CAS number: 85-00-7
Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 Feb 1998 to 09 Feb 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: EPA 123-2 (Growth and reproduction of aquatic plants)
- Deviations:
- not specified
- GLP compliance:
- yes
- Analytical monitoring:
- yes
- Details on sampling:
- At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels. At the end of the test the blank solutions were sampled and analysed in the same manner.
- Vehicle:
- no
- Details on test solutions:
- The test substance was soluble in water at a higher concentration than the test concentrations selected. Therefore, the test solutions were prepared without the use of an organic solvent. A 1000 mL stock solution (nominal concentration 10000 µg/L) was prepared by the direct addition of the test substance to sterile culture medium. The highest nominal concentration test solution was prepared, using sterile medium, by the addition of an aliquot of this stock. All lower test concentrations were similarly prepared, using aliquots of the nominal 80 µg/L test solution. The control consisted of culture medium only. After preparation, a visual assessment of the test solutions showed them all to be clear and colourless. Using aseptic techniques, 100 mL volumes of the appropriate test solution were dispensed to each test replicate vessel, but blank solution volumes were increased to 750 mL, to provide sufficient volume for chemical analyses. The remainder of the test solutions were used for physical and chemical analyses.
- Test organisms (species):
- Navicula pelliculosa
- Details on test organisms:
- TEST ORGANISM
- Common name: Diatom
- Strain: UTEX 667
- Age of inoculum (at test initiation): Exponential growth phase
- Culturing media and conditions: same - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 120 h
- Test temperature:
- 24 ± 2°C
- pH:
- 7.6 - 9.0
- Nominal and measured concentrations:
- - Nominal concentration: 0 (negative control), 0.65, 1.3, 2.5, 5.0, 10, 20, 40 and 80 µg test materail/L
- Measured concentration: < LOQ (negative control), 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg test material/L, respectively. See Table 1 in 'Any other information on materials and methods inlc. tables'. - Details on test conditions:
- TEST SYSTEM
- Test vessel: Borosilicate glass conical flasks
- Vessel capacity: 250 mL
- Type: Closed with polyurethane foam bungs
- Volume of test solution: 100 mL
- Volume of blank solution: 750 mL
- Shaking: Yes, at 140 rpm
- Initial cells density: 0.3E+04 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- Vessel position: Randomised by rows, and re-randomised daily
GROWTH MEDIUM
- Standard medium used: Yes
WATER PARAMETERS
- pH: The pH of each test solution was measured at the start of the test, using the excess remaining after filling the test vessels. At the end of the test the pH of two of the replicate test solutions (containing algae) from the culture medium control and each test concentration was determined.
- Temperatrue: The temperature of the incubator was measured daily by a thermometer and contimuously monitored with hourly recording of values, using an automatic recording system linked to a thermistor.
OTHER TEST CONDITIONS
- Light type: 4480 lux (test incubator) and 4200 lux (analytical blank incubator), "cool-white" illumination, (measured once during the study).
- Photoperiod: Continuously
EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: After 1 , 2, 3, 4 and 5 days (24, 48, 72, 96 and 120 hours), samples were removed from each test replicate and the appropriate blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density. - Reference substance (positive control):
- no
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 1.99 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: pure test substance
- Basis for effect:
- growth rate
- Remarks on result:
- other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 2.3 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: pure test substance
- Basis for effect:
- growth rate
- Remarks on result:
- other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 6 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: test substance cation species
- Basis for effect:
- growth rate
- Remarks on result:
- other: Original value presented in study
- Remarks:
- 95% C.L.: 5.4 - 6.7 µg/L
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 5.2 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- other: test substance cation species
- Basis for effect:
- growth rate
- Remarks on result:
- other: Original value presented in study
- Details on results:
- Overview of the results are provided in Table 2 and Table 4 at ‘Any other information on results incl. tables’
DENSITY
At the start of the test, the initial algal density was 0.326E+04 cells/mL. Following 72-hours of exposure, cell density in the control averaged as 5.64E+06 cells/mL. The 72-hour averaged cell density in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels were 10.7,11.2, 7.43, 3.83, 2.44, 0.433, 0.276 and 0.111E+04 cells/mL, respectively. After 120 hours treatment, the cell density in the control averaged as 9.99E+05 cells/mL. The average cell density in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels were 13.1, 15.7, 12.2, 4.45, 1.31, 0.0569, 0.0234 and 0.0076E+05 cells/mL
BIOMASS
The biomass integrals were determined by the areas under the growth curves of algal cultures with different concentrations of the test item. Following 72-hours of exposure, mean area under the growth curve in the control was 3.3. The 72-hour mean area under the growth curve in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels were 6.7, 6.6, 4.3, 2.0, -0.1, -0.2 and -0.5, respectively. At 72 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 5.2 µg/L. The 72-hour EbC50 was determined to be 4.2 µg/L (corresponding with 95% confidence interval 3.8 – 4.7 µg/L). Following 120-hours of exposure, mean area under the growth curve in the control was 82.2. The 120-hour mean area under the growth curve in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels 122.5, 140.0, 102.1, 39.5, 13.9, 0.2, -0.3 and -0.9, respectively. At 120 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 2.9, 5.2, 11, 28 and 62µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 1.0 µg/L. The 120-hour EbC50 was determined to be 2.9 µg/L (corresponding with 95% confidence interval 2.6 – 3.2 µg/L).
GROWTH RATE
Following 72 hours of exposure, growth rate in the control averaged 0.950 days-1. The 72-hour growth rate in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels averaged 1.165, 1.178, 1.042, 0.821, 0.670, 0.095, -0.055, and -0.359 days-1, respectively. At 72 hours, a significant reduction in growth rate was detected in the 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 5.2 µg/L. The 72-hour ErC50 was determined to be 6.0 µg/L (corresponding with 95% confidence interval 5.4 – 6.7 µg/L). Following 120 hours of exposure, growth rate in the control averaged 1.145 days-1. The 120-hour growth rate in the 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg/L treatment levels averaged 1.200, 1.235, 1.185, 0.983, 0.739, 0.112, -0.066, and -0.291 days-1, respectively. At 120 hours, a significant reduction in growth rate was detected in the 5.2, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 2.9 µg/L. The 120-hour ErC50 was determined to be 5.8 µg/L (corresponding with 95% confidence interval 5.2 – 6.4 µg/L). - Reported statistics and error estimates:
- The area under the growth curve of the algae were examined by one-way analysis of variance, and Dunnett’s procedure was used to identify significant differences (P = 0.05) from the culture medium control.
- Validity criteria fulfilled:
- yes
- Conclusions:
- Based on the findings, the 72-hour NOErC was determined to be 5.2 µg test material/L, equivalent to 1.99 µg test substance/L. The 72-hour ErC50 was determined to be 6.0 µg test material/L (95% confidence interval 5.4 – 6.7 µg/L). This 72-hour ErC50 value equivalent to 2.30 µg pure test substance/L.
- Executive summary:
The influence of the test substance on the growth of a freshwater diatom, Navicula pelliculosa, was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 and in compliance with GLP criteria. The diatoms were exposed to measured concentrations of 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg test material/L (measured by HPLC). In addition, a negative control was included in this study as well. The test was carried out in an incubator with a temperature range of 24 ± 2 °C. Flasks were shaken at a speed of 140 rpm. Illumination of 4200 - 4480 lux was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each day to minimize spatial differences in the incubation. The pH of the test started from a range of 8.6 – 9.0 and ended with a range of 7.6 – 7.8. Biomass of the algae was determined by cell counts everyday using a coulter counter.
At the start of the test, the initial algal density was 0.326E+04 cells/mL. Following 72-hours of exposure, the average cell density in the control reached 5.64E+06 cells/mL. The biomass integrals for the treated algal curltures were determined by the areas under the growth curves. Following 72-hours of exposure, mean area under the growth curve in the control was 3.3. At 72 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 5.2 µg test material/L. The 72-hour EbC50 was determined to be 4.2 µg test material/L (with corresponding 95% confidence interval 3.8 – 4.7 µg/L).
Following 120-hours of exposure, mean area under the growth curve in the control was 82.2. At 120 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 2.9, 5.2, 11, 28 and 62µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 1.0 µg/L. The 120-hour EbC50 was determined to be 2.9 µg/L (with corresponding 95% confidence interval 2.6 – 3.2 µg/L).
Following 72 hours of exposure, the growth rate in the control averaged 0.950 days-1. At 72 hours, a significant reduction in growth rate was detected in the 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 5.2 µg/L(equivalent to 1.99 µg pure test substance/L ). The 72-hour ErC50 was determined to be 6.0 µg/L (corresponding with 95% confidence interval 5.4 – 6.7 µg/L).This 72-hour ErC50 value equivalent to 2.30 µg pure test substance/L.
Following 120 hours of exposure, growth rate in the control averaged 1.145 days-1.
At 120 hours, a significant reduction in growth rate was detected in the 5.2, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 2.9 µg test material/L. The 120-hour ErC50 was determined to be 5.8 µg test material/L (corresponding with 95% confidence interval 5.2 – 6.4 µg/L).
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 Jan 1998 to 19 Jan 1998
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: EPA 123-2 (Growth and reproduction of aquatic plants)
- Version / remarks:
- 1982
- GLP compliance:
- yes
- Analytical monitoring:
- yes
- Details on sampling:
- At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels. At the end of the test each blank solution was sampled and analysed in the same manner.
- Vehicle:
- no
- Details on test solutions:
- This study was run with a culture medium control together with nominal concentrate concentrations of 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg test material/L. The test substance is soluble in water at a higher concentration than the test concentrations selected. Therefore, the test solutions were prepared without the use of an organic solvent. A 2000 mL volume of the highest nominal concentration test solution (180 mg/L) was prepared by the direct addition of the test substance concentrate to sterile culture medium. The lower test concentrations were prepared, using sterile culture medium, by the addition of aliquots of the nominal 180 mg/L test solution, to final volumes of 1000 mL. The control consisted of culture medium only. After preparation, a visual assessment of the test solutions showed them all to be clear. A brown colouration, deepening with increasing nominal concentration, was apparent from 12 mg/L upwards. Using aseptic techniques, 100 mL volumes of the appropriate test solution were dispensed to each test and blank vessel, with the remainder of the test solutions being used for physical and chemical analyses.
- Test organisms (species):
- Skeletonema costatum
- Details on test organisms:
- TEST ORGANISM
- Strain: CCAP 1077/IC
- Source: Cultured in laboratory under axenic condition
- Age of inoculum: A 2 day old culture of the algae (in the growth phase) was used as inoculum for the test.
- Method of cultivation: The culture was grown in the medium, and under the same environmental conditions as the test. - Test type:
- static
- Water media type:
- saltwater
- Limit test:
- no
- Total exposure duration:
- 120 h
- Test temperature:
- 20 ± 2°C
- pH:
- 8.0 - 8.7
- Salinity:
- 31.6‰ ( measured post filter sterilisation)
- Nominal and measured concentrations:
- - Nominal concentration: 0 (negative control), 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg test material/L
- Measured concentration: < LOQ (negative control), 1.4, 2.8, 5.6, 11, 22. 47, 93 and 190 mg test material/L, respectively. (See Table 1 in 'Any other information on materials and methods incl. tables'). - Details on test conditions:
- TEST SYSTEM
- Test vessel: 250 mL borosilicate glass conical flasks
- Type: Closed with polyurethane foam bungs
- Filled volume: 100 mL of test solution
- Shaking: Yes, 100 rpm
- Initial cells density: 1.0E+04 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6
The positions of the test vessels in the incubator were randomised by rows, and re-randomised daily. One blank vessel (without algal inoculum) for the culture medium control and each test concentration was incubated concurrently.
GROWTH MEDIUM
- Standard medium used: yes
WATER PARAMETERS
- Light intensity: The light intensity was measured once during the study.
- pH: The pH of each test solution was measured at the start of the test, using the excess remaining after filling the test vessels. At the end of the test the pH of two of the replicate test solutions (containing algae) from the culture medium control and each test concentration was determined.
- Temperature: The temperature of the incubator was measured daily by a thermometer calibrated to 0.1°C . Also, the temperature was continuously monitored with a hourly recording of values, using an automatic recording system linked to a thermistor.
- Salinity: The salinity of excess culture medium was measured at the start of the test
OTHER TEST CONDITIONS
- Photoperiod: 16 hour light and 8 hour dark (“on” daily 05.00 - 21.00 hour)
- Light intensity: 4440 lux (52.1 µ einsteins/m2/s)
- Light resource: “cool white” light
EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell density of the culture used as the inoculum for the test was determined by microscope counting using a haemocytometer chamber.
- Other: The particle density of the inoculum culture was also determined by coulter counter and was 0.541E+06 particles/mL, equivalent to
2.10 cells per particle, with a nominal inoculum level of 0.476E+04 particles/mL. This was consistent with the haemocytometer observation that the culture contained predominantly 1, 2 and 3-cell “chains”. Three 100 mL volumes of coulter electrolyte, inoculated in the same manner, had a mean measured particle density of 0.462E+04 particles/mL. This value was used as the inoculum level in the growth calculations. All subsequent density determinations were carried out by electronic particle counting using a coulter counter, counting at a lower threshold equivalent spherical diameter of approximately 2.9 µm. After 1, 2, 3, 4 and 5 days (24, 48, 72, 96 and 120 hours), samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the particle density. - Reference substance (positive control):
- no
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 8.81 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- other: pure test substance
- Basis for effect:
- growth rate
- Remarks on result:
- other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 21.83 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- other: pure test substance
- Basis for effect:
- growth rate
- Remarks on result:
- other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 57 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- other: test substance cation species
- Basis for effect:
- growth rate
- Remarks on result:
- other: Original value presented in study
- Remarks:
- 95% C.L.: 51 - 65 mg/L
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 23 mg/L
- Nominal / measured:
- meas. (initial)
- Conc. based on:
- other: test substance cation species
- Basis for effect:
- growth rate
- Remarks on result:
- other: Original value presented in study
- Details on results:
- Overview of the results are provided in Table 2 and Table 4 at ‘Any other information on results incl. tables’.
DENSITY
At the start of the test, the initial algal density was 1.0E+04 cells/mL. Following 72-hours of exposure, cell density in the control averaged as 3.05E+05 cells/mL. The 72-hour averaged cell density in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels were 2.59, 3.02, 2.75, 2.83, 2.61, 1.45, 0.108 and 0.0347E+05 cells/mL, respectively. After 120 hours treatment, the cell density in the control averaged as 8.41E+05 cells/mL. The average cell density in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels were 7.57, 7.54, 7.30, 7.86, 5.81, 6.24, 0.116 and 0.0495E+05 cells/mL.
BIOMASS
The biomass integrals were determined by the areas under the growth curves of algal cultures with different concentrations of the test item. Following 72-hours of exposure, mean area under the growth curve in the control was 25.4. The 72-hour mean area under the growth curve in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels were 23.1, 23.4, 20.5, 21.5, 18.7, 12.0, 0.9 and -0.1, respectively. At 72 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 3.0 mg/L. The 72-hour EbC50 was determined to be 26 mg/L (corresponding with 95% confidence interval 23 – 31 mg/L). Following 120-hours of exposure, mean area under the growth curve in the control was 132.8. The 120-hour mean area under the growth curve in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels 123.5, 124.6, 18.7, 127.6, 108.9, 91.2, 2.1 and -0.2, respectively. At 120 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 3.0 mg/L. The 120-hour EbC50 was determined to be 37 mg/L (corresponding with 95% confidence interval 32 – 43 mg/L).
GROWTH RATE
Following 72 hours of exposure, growth rate in the control averaged 1.397 days-1. The 72-hour growth rate in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels averaged 1.342, 1.393, 1.362, 1.371, 1.345, 1.189, 0.207 and -0.095 days-1, respectively. At 72 hours, a significant reduction in growth rate was detected in the 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 23 mg/L. The 72-hour ErC50 was determined to be 57 mg/L (corresponding with 95% confidence interval 51 – 65 mg/L). Following 120 hours of exposure, growth rate in the control averaged 1.041 days-1. The 120-hour growth rate in the 1.5, 3.0, 6.0, 12, 23, 45, 90 and 180 mg/L treatment levels averaged 1.020, 1.019, 1.013, 1.027, 0.967, 0.981, 0.184 and 0.014 days-1, respectively. At 120 hours, a significant reduction in growth rate was detected in the 23, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 12 mg/L. The 120-hour ErC50 was determined to be 61 mg/L (corresponding with 95% confidence interval 54 – 70 mg/L). - Validity criteria fulfilled:
- yes
- Conclusions:
- The 72-hour NOErC was determined to be 23 mg/L, equivalent to 8.81 mg test substance/L) and the 72-hour ErC50 was determined to be 57 mg/L (with 95% confidence limits of 62 - 74 mg/L). The 72-hour ErC50 equivalent to 21.83 mg pure test substance /L.
- Executive summary:
The influence of the test substance on the growth of marine algae, Skeletonema costatum, was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 and in compliance with GLP criteria. The algae were exposed to concentrations measured by HPLC of 1.4, 2.8, 5.6, 11, 22, 47, 93 and 190 mg/L. In addition, a negative control was included in this study as well. The test was carried out in an incubator with a temperature range of 20 ± 2°C. Flasks were shaken at a speed of 100 rpm. Illumination of 4440 lux was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each day to minimize spatial differences in the incubation. The pH of the test started from a range of 8.0 – 8.1 and ended with a range of 8.1 – 8.7. Biomass of the algae was determined by cell counts everyday using a coulter counter.
At the start of the test, the initial algal density was 1.0E+04 cells/mL. Following 72 and 120 hours of exposure, the average cell density in the control reached 3.05E+05 cells/mL and 8.41E+05 cells/mL, respectively. The biomass integrals were determined by the areas under the growth curves of algal cultures espoxed to different concentrations of the test item.
Following 72-hours of exposure, mean area under the growth curve in the control was 25.4. At 72 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 3.0 mg/L. The 72-hour EbC50 was determined to be 26 mg/L (corresponding with 95% confidence interval 23 – 31 mg/L).
Following 120-hours of exposure, mean area under the growth curve in the control was 132.8. At 120 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 3.0 mg test material/L. The 120-hour EbC50 was determined to be 37 mg/L (corresponding with 95% confidence interval 32 – 43 mg/L).
Following 72 hours of exposure, growth rate in the control averaged 1.397 days-1. At 72 hours, a significant reduction in growth rate was detected in the 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 23 mg/L, equivalent to 8.81 mg pure test substance/L). The 72-hour ErC50 was determined to be 57 mg/L (corresponding with 95% confidence interval 51 – 65 mg/L). The 72-hour ErC50 equivalent to 21.83 mg pure test substance /L.
Following 120 hours of exposure, growth rate in the control averaged 1.041 days-1. At 120 hours, a significant reduction in growth rate was detected in the 23, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 12 mg test material/L. The 120-hour ErC50 was determined to be 61 mg/L (corresponding with 95% confidence interval 54 – 70 mg/L).
Referenceopen allclose all
Table 2. Algae cell density
Nominal concentration test material (µg/L) | Mean measured concentration test material (µg/L) | Cell density (x1.0E+04 cells/mL) | |||||
Replicate | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | ||
Culture medium control | A | 0.192 | 1.1 | 4.77 | 20.7 | 59.3 | |
B | 0.220 | 1.1 | 5.62 | 31.2 | 148.0 | ||
C | 0.093 | 0.808 | 3.92 | 19.5 | 70.0 | ||
D | 0.127 | 1.1 | 5.86 | 25.3 | 81.1 | ||
E | 0.152 | 1.6 | 8.32 | 40.2 | 145.0 | ||
F | 0.213 | 1.2 | 5.35 | 23.6 | 95.9 | ||
Mean | 0.166 | 1.1 | 5.64 | 26.8 | 99.9 | ||
0.65 | 0.3 | A | 0.232 | 1.9 | 10.7 | 48.2 | 133 |
B | 0.208 | 1.7 | 9.8 | 42.0 | 128 | ||
C | 0.250 | 2.3 | 11.7 | 45.9 | 133 | ||
Mean | 0.230 | 1.96 | 10.7 | 45.4 | 131 | ||
1.3 | 0.56 | A | 0.244 | 2.03 | 10.8 | 46.4 | 165 |
B | 0.163 | 1.62 | 11.9 | 50.8 | 143 | ||
C | 0.113 | 1.44 | 10.8 | 53.0 | 162 | ||
Mean | 0.173 | 1.70 | 11.2 | 50.1 | 157 | ||
2.5 | 1 | A | 0.074 | 1.1 | 6.62 | 30.3 | 122 |
B | 0.11 | 1.31 | 7.84 | 37.3 | 132 | ||
C | 0.094 | 1.42 | 7.82 | 33.6 | 112 | ||
Mean | 0.093 | 1.28 | 7.43 | 33.7 | 122 | ||
5 | 2.9 | A | 0.118 | 0.98 | 5.82 | 22.0 | 70.7 |
B | 0.109 | 0.613 | 1.54 | 4.8 | 13.9 | ||
C | 0.064 | 0.791 | 4.12 | 15.0 | 49.0 | ||
Mean | 0.097 | 0.794 | 3.83 | 13.9 | 44.5 | ||
10 | 5.2 | A | 0.218 | 1.12 | 2.34 | 5.36 | 15.40 |
B | 0.228 | 1.51 | 2.92 | 6.17 | 17.00 | ||
C | 0.164 | 1.18 | 2.05 | 3.18 | 6.96 | ||
Mean | 0.203 | 1.27 | 2.44 | 4.90 | 13.1 | ||
20 | 11 | A | 0.043 | 0.203 | 0.301 | 0.276 | 0.250 |
B | 0.197 | 0.635 | 0.836 | 0.954 | 1.15 | ||
C | 0.035 | 0.245 | 0.163 | 0.226 | 0.308 | ||
Mean | 0.092 | 0.361 | 0.433 | 0.485 | 0.569 | ||
40 | 28 | A | 0.086 | 0.467 | 0.47 | 0.55 | 0.418 |
B | 0.036 | 0.355 | 0.138 | 0.148 | 0.106 | ||
C | 0.061 | 0.42 | 0.221 | 0.316 | 0.178 | ||
Mean | 0.061 | 0.414 | 0.276 | 0.338 | 0.234 | ||
80 | 62 | A | 0.006 | 0.211 | 0.168 | 0.226 | 0.128 |
B | 0.001 | 0.222 | 0.089 | 0.118 | 0.07 | ||
C | 0.001 | 0.259 | 0.076 | 0.092 | 0.03 | ||
Mean | 0.003 | 0.231 | 0.111 | 0.145 | 0.076 | ||
Inoculum (Day 0) cell density = 0.326E+04 cells/mL
NB. Values of 0.001 (x1.0E+04 cells/ml ) were recorded where the corresponding blank particle count exceeded that of the test algal solutions.
Table 3. Results of the area under the growth curve
Nominal concentration test material (µg/L) | Mean measured concentration test material (µg/L) | 0 - 3 day | 0 - 4 day | 0 - 5 day | |||
Mean | Percentage | Mean | Percentage | Mean | Percentage | ||
Culture medium control | - | 3.3 | - | 19.2 | - | 82.2 | - |
0.65 | 0.30 | 6.7* | 204 | 34.5* | 180 | 122.5* | 149 |
1.3 | 0.56 | 6.6* | 201 | 36.9* | 193 | 140.0* | 170 |
2.5 | 1.00 | 4.3 | 129 | 24.5 | 128 | 102.1 | 124 |
5 | 2.9 | 2 | 60 | 10.5* | 55 | 39.5* | 48 |
10 | 5.2 | 1.9 | 57 | 5.2* | 27 | 13.9* | 17 |
20 | 11 | -0.1* | -4 | 0.0* | 0 | 0.2* | 0 |
40 | 28 | -0.2* | -6 | -0.2* | -1 | -0.3* | 0 |
80 | 62 | -0.5* | -16 | -0.7* | -4 | -0.9* | -1 |
* Significant difference (P=0.05) from the culture medium control
Table 4. Results of growth rate
Nominal concentration test material (µg/L) | Mean measured concentration test material (µg/L) | 0 - 3 day | 0 - 4 day | 0 - 5 day | |||
Mean | Percentage | Mean | Percentage | Mean | Percentage | ||
Culture medium control | 0.95 | 1.102 | 1.145 | ||||
0.65 | 0.3 | 1.165 | 123 | 1.234 | 112 | 1.2 | 105 |
1.3 | 0.56 | 1.178 | 124 | 1.259 | 114 | 1.235 | 108 |
2.5 | 1 | 1.042 | 110 | 1.16 | 105 | 1.185 | 103 |
5 | 2.9 | 0.821 | 86 | 0.939 | 85 | 0.983 | 86 |
10 | 5.2 | 0.67 | 71 | 0.678* | 62 | 0.739* | 65 |
20 | 11 | 0.095* | 10 | 0.099* | 9 | 0.112* | 10 |
40 | 28 | -0.055* | -6 | 0.009* | 1 | -0.066* | -6 |
80 | 62 | -0.359* | -38 | -0.202* | -18 | -0.291* | -25 |
* Significant difference (P=0.05) from the culture medium control
Calculation of key result
The original effect levels were expressed test material, an aqueous concentrate of the test substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species) and corrected for the amount of water:
72-hour ErC50: 20.5% x 1.868 x 6.0 µg/L= 2.30 µg/L
72-hour NOErC: 20.5% x 1.868 x 5.2 µg/L= 1.99 µg/L
Table 2. Algal density
Nominal concentration (mg/L) | Replicate | Algal particle density (x1.0E+04 particles/mL) | ||||
Day 1 | Day 2 | Day 3 | Day 4 | Day 5 | ||
Culture | A | 1.19 | 9.98 | 36.1 | 53.7 | 91.2 |
B | 1.26 | 9.4 | 34.7 | 52.8 | 86.4 | |
C | 1.11 | 10.8 | 30.7 | 49.1 | 83.4 | |
D | 1.1 | 9.68 | 26 | 49 | 94.4 | |
E | 0.966 | 11 | 28.5 | 46.9 | 78.5 | |
F | 0.887 | 10.2 | 27.1 | 54.8 | 70.7 | |
Mean | 1.09 | 10.2 | 30.5 | 51.1 | 84.1 | |
1.5 | A | 0.906 | 10.4 | 27.7 | 48.7 | 68.5 |
B | 0.96 | 10.1 | 25.3 | 53.2 | 80.6 | |
C | 0.875 | 10.7 | 24.6 | 49.6 | 78.1 | |
Mean | 0.914 | 10.4 | 25.9 | 50.5 | 75.7 | |
3 | A | 0.922 | 9.41 | 30 | 49.8 | 77.1 |
B | 1.26 | 8.2 | 33.5 | 49.2 | 79.4 | |
C | 0.998 | 7.72 | 27.1 | 48.8 | 69.6 | |
Mean | 1.06 | 8.44 | 30.2 | 49.3 | 75.4 | |
6 | A | 1.11 | 6.77 | 28 | 48.9 | 75.2 |
B | 1.04 | 7.56 | 30.1 | 48.8 | 74.4 | |
C | 1.06 | 6.31 | 24.3 | 48.8 | 69.4 | |
Mean | 1.07 | 6.88 | 27.5 | 48.8 | 73 | |
12 | A | 2.1 | 6.89 | 31.7 | 52 | 92 |
B | 1.91 | 6.11 | 25.9 | 54.4 | 69 | |
C | 1.96 | 6.54 | 27.2 | 54.4 | 74.7 | |
Mean | 1.99 | 6.51 | 28.3 | 53.6 | 78.6 | |
23 | A | 1.04 | 5.94 | 28.3 | 50.1 | 59 |
B | 1.09 | 5.97 | 26.3 | 48.1 | 58.4 | |
C | 0.987 | 5.25 | 23.8 | 48.9 | 56.8 | |
Mean | 1.04 | 5.72 | 26.1 | 49 | 58.1 | |
45 | A | 1.04 | 4.82 | 19.4 | 47.1 | 67 |
B | 1.14 | 3.37 | 15.2 | 37.8 | 64.3 | |
C | 1.01 | 3.59 | 14.5 | 37.1 | 56 | |
Mean | 1.06 | 3.93 | 16.4 | 40.7 | 62.4 | |
90 | A | 0.706 | 0.743 | 0.898 | 1.11 | 1.56 |
B | 0.603 | 0.934 | 0.602 | 0.766 | 1.01 | |
C | 0.828 | 1.10 | 1.08 | 1.57 | 0.906 | |
Mean | 0.712 | 0.926 | 0.86 | 1.15 | 1.16 | |
180 | A | 0.485 | 0.427 | 0.344 | 0.658 | 0.582 |
B | 0.463 | 0.352 | 0.29 | 0.376 | 0.566 | |
C | 0.487 | 0.325 | 0.408 | 0.32 | 0.338 | |
Mean | 0.478 | 0.368 | 0.347 | 0.451 | 0.495 | |
Note: Inoculum (Day 0) particle density = 0.462E+04 particles/mL
Table 3. Results of mean area under the growth curve
Nominal concentration (mg/L) | 0 - 3 day | 0 - 4 day | 0 - 5 day | |||
Mean area under growth curve | Percentrage of cultural medium control | Mean area under growth curve | Percentrage of cultural medium control | Mean area under growth curve | Percentrage of cultural medium control | |
Culture medium control | 25.4 | 65.7 | 132.8 | |||
1.5 | 23.1 | 91 | 60.8 | 93 | 123.5 | 93 |
3.0 | 23.4 | 92 | 62.7 | 95 | 124.6 | 94 |
6.0 | 20.5* | 81 | 58.2 | 89 | 118.7* | 89 |
12 | 21.5* | 85 | 62 | 94 | 127.6 | 96 |
23 | 18.7* | 74 | 55.8* | 85 | 108.9* | 82 |
45 | 12.0* | 47 | 40.1* | 61 | 91.2* | 69 |
90 | 0.9* | 4 | 1.5* | 2 | 2.1* | 2 |
180 | -0.1* | 0 | -0.2* | 0 | -0.2* | 0 |
* Significant difference (P=0.05) from the culture medium control
Table 4. Results of mean growth rate
Nominal concentration (mg/L) | 0 - 3 day | 0 - 4 day | 0 - 5 day | |||
Mean growth rate | Percentrage of cultural medium control | Mean growth rate | Percentrage of cultural medium control | Mean growth rate | Percentrage of cultural medium control | |
Culture medium control | 1.397 | 1.176 | 1.041 | |||
1.5 | 1.342 | 96 | 1.174 | 100 | 1.02 | 98 |
3 | 1.393 | 100 | 1.167 | 99 | 1.019 | 98 |
6 | 1.362 | 97 | 1.165 | 99 | 1.013 | 97 |
12 | 1.371 | 98 | 1.188 | 101 | 1.027 | 99 |
23 | 1.345 | 96 | 1.166 | 99 | 0.967* | 93 |
45 | 1.189* | 85 | 1.119 | 95 | 0.981 | 94 |
90 | 0.207* | 15 | 0.228* | 19 | 0.184* | 18 |
180 | -0.095* | 0 | -0.006* | 0 | 0.014* | 1 |
* Significant difference (P=0.05) from the culture medium control
Calculation of key result
The original effect levels were expressed as test material, an aqueous concentrate of the test substance.The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of test substance divided by 184.2 g/mol molecular weight of cation species) and corrected for the amount of water.:
72-hour ErC50: 20.5% x 1.868 x 57 mg/L= 21.83 mg/L
72-hour NOErC: 20.5% x 1.868 x 23 mg/L= 8.81 mg/L
Description of key information
Freshwater, 72-h NOErC = 1.99 µg test substance/L, Navicula pelliculosa, EPA 123 -2, Smyth 1998
Freshwater, 72-h ErC50 = 2.30 µg test substance /L, Navicula pelliculosa, EPA 123 -2, Smyth 1998
Marine water, 72-h NOErC = 8.81 mg test substance/L, Skeletonema costatum, EPA 123 -2, Smyth 1998
Marine water, 72-h ErC50 = 21.83 mg test substance/L, Skeletonema costatum, EPA 123 -2, Smyth 1998
Key value for chemical safety assessment
- EC50 for freshwater algae:
- 2.3 µg/L
- EC50 for marine water algae:
- 21.83 mg/L
- EC10 or NOEC for freshwater algae:
- 1.99 µg/L
- EC10 or NOEC for marine water algae:
- 8.81 mg/L
Additional information
Freshwater
The influence of the test substance on the growth of a freshwater diatom, Navicula pelliculosa, was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 and in compliance with GLP criteria. The diatoms were exposed to measured concentrations of 0.30, 0.56, 1.0, 2.9, 5.2, 11, 28 and 62 µg test material/L (measured by HPLC). In addition, a negative control was included in this study as well. The test was carried out in an incubator with a temperature range of 24 ± 2 °C. Flasks were shaken at a speed of 140 rpm. Illumination of 4200 - 4480 lux was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each day to minimize spatial differences in the incubation. The pH of the test started from a range of 8.6 – 9.0 and ended with a range of 7.6 – 7.8. Biomass of the algae was determined by cell counts everyday using a coulter counter.
At the start of the test, the initial algal density was 0.326E+04 cells/mL. Following 72-hours of exposure, the average cell density in the control reached 5.64E+06 cells/mL. The biomass integrals for the treated algal curltures were determined by the areas under the growth curves. Following 72-hours of exposure, mean area under the growth curve in the control was 3.3. At 72 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 5.2 µg test material/L. The 72-hour EbC50 was determined to be 4.2 µg test material/L (with corresponding 95% confidence interval 3.8 – 4.7 µg/L).
Following 120-hours of exposure, mean area under the growth curve in the control was 82.2. At 120 hours, a significant reduction in biomass integrals was detected in the 0.30, 0.56, 2.9, 5.2, 11, 28 and 62µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 1.0 µg/L. The 120-hour EbC50 was determined to be 2.9 µg/L (with corresponding 95% confidence interval 2.6 – 3.2 µg/L).
Following 72 hours of exposure, the growth rate in the control averaged 0.950 days-1. At 72 hours, a significant reduction in growth rate was detected in the 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 5.2 µg/L(equivalent to 1.99 µg pure test substance/L ). The 72-hour ErC50 was determined to be 6.0 µg/L (corresponding with 95% confidence interval 5.4 – 6.7 µg/L).This 72-hour ErC50 value equivalent to 2.30 µg pure test substance/L.
Following 120 hours of exposure, growth rate in the control averaged 1.145 days-1.
At 120 hours, a significant reduction in growth rate was detected in the 5.2, 11, 28 and 62 µg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 2.9 µg test material/L. The 120-hour ErC50 was determined to be 5.8 µg test material/L (corresponding with 95% confidence interval 5.2 – 6.4 µg/L).
Marine water
The influence of the test substance on the growth of marine algae, Skeletonema costatum, was investigated in a 5-day static test in accordance with EPA FIFRA TG 123-2 and in compliance with GLP criteria. The algae were exposed to concentrations measured by HPLC of 1.4, 2.8, 5.6, 11, 22, 47, 93 and 190 mg/L. In addition, a negative control was included in this study as well. The test was carried out in an incubator with a temperature range of 20 ± 2°C. Flasks were shaken at a speed of 100 rpm. Illumination of 4440 lux was provided by overhead cool-white continuous fluorescent lights. Flasks were randomly repositioned each day to minimize spatial differences in the incubation. The pH of the test started from a range of 8.0 – 8.1 and ended with a range of 8.1 – 8.7. Biomass of the algae was determined by cell counts everyday using a coulter counter.
At the start of the test, the initial algal density was 1.0E+04 cells/mL. Following 72 and 120 hours of exposure, the average cell density in the control reached 3.05E+05 cells/mL and 8.41E+05 cells/mL, respectively. The biomass integrals were determined by the areas under the growth curves of algal cultures espoxed to different concentrations of the test item.
Following 72-hours of exposure, mean area under the growth curve in the control was 25.4. At 72 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOEbC was determined to be 3.0 mg/L. The 72-hour EbC50 was determined to be 26 mg/L (corresponding with 95% confidence interval 23 – 31 mg/L).
Following 120-hours of exposure, mean area under the growth curve in the control was 132.8. At 120 hours, a significant reduction in biomass integrals was detected in the 6, 12, 23, 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOEbC was determined to be 3.0 mg test material/L. The 120-hour EbC50 was determined to be 37 mg/L (corresponding with 95% confidence interval 32 – 43 mg/L).
Following 72 hours of exposure, growth rate in the control averaged 1.397 days-1. At 72 hours, a significant reduction in growth rate was detected in the 45, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 72-hour NOErC was determined to be 23 mg/L, equivalent to 8.81 mg pure test substance/L). The 72-hour ErC50 was determined to be 57 mg/L (corresponding with 95% confidence interval 51 – 65 mg/L). The 72-hour ErC50 equivalent to 21.83 mg pure test substance /L.
Following 120 hours of exposure, growth rate in the control averaged 1.041 days-1. At 120 hours, a significant reduction in growth rate was detected in the 23, 90 and 180 mg/L treatment levels, compared to the control data. Therefore, the 120-hour NOErC was determined to be 12 mg test material/L. The 120-hour ErC50 was determined to be 61 mg/L (corresponding with 95% confidence interval 54 – 70 mg/L).
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