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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-01-28 to 2019-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006 / Annex 5 corrected 28 July 2011.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Purity: 99.7%
Batch No.: 80031745a
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 1.8 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 100 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 1.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium. The pH of the stock solution was adjusted from 5.3 to 6.4 using 1 M NaOH solution (Merck, Darmstadt, Germany). The pH of the test medium was adjusted from 8.3 to 6.5 using 1 M HCL solution (Merck, Darmstadt, Germany) in order to match the pH of the stock. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL. Any residual volumes were discarded
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test System
Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3 500 mg/L, K2HPO4 39.5 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3 20 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L, (NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L, NaHCO3 50 mg/L, Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L), pH 8.1 ± 0.2 4
Test type:
static
Water media type:
other: Milli-RO water (tap Final Report Page 11 Test Facility Study No. 20176452 water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
Limit test:
yes
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
21-24°C
pH:
8.1 ± 0.2
Nominal and measured concentrations:
0.10, 1.0, 10 and 100 mg/L
Details on test conditions:
Preparation of Test Solutions
The batch of 1,4-Benzodioxan-2-carboxylic acid tested was an off-white to brown solid with a purity of 99.7% and was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in medium. The pH of the stock solution was adjusted from 5.3 to 6.4 using 1 M NaOH solution (Merck, Darmstadt, Germany). The pH of the test medium was adjusted from 8.3 to 6.5 using 1 M HCL solution (Merck, Darmstadt, Germany) in order to match the pH of the stock. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test System
Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh Water Algae Culture
Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3 500 mg/L, K2HPO4 39.5 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3 20 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L, (NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use. Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L, NaHCO3 50 mg/L, Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L), pH 8.1 ± 0.2


Test Concentrations:
Test item: 0.10, 1.0, 10 and 100 mg/L
Control: Test medium without test item or other additives.
Replicates: 6 replicates of the control and highest test concentration, 3 replicates each of lower concentrations, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test Medium: M2
Cell density: An initial cell density of 1E+04 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 87 to 91 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report.
Frequency: at t=0 h, t=24 h and t=72 h.
Volume: 1.8 mL from the approximate centre of the test vessels.
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 100 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 1.8 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Measurements and Recordings
pH: At the beginning and at the end of the test, for the limit concentration and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the test, microscopic observations were performed on the test concentration of nominally 100 mg/L to observe for any abnormal appearance of the algae compared to the control.

Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Lower concentrations were measured only at the end of the exposure period.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 100 mg/L when compared to the control.
Results with reference substance (positive control):
The 72h-EC50 for growth rate inhibition (ERC50) was 1.30 mg/L with a 95% confidence interval ranging from 1.29 to 1.32 mg/L. The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years. In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at Charles River Den Bosch.
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of the test item. The 72h-EC50 for growth rate (ERC50) and yield (EYC50) inhibition was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L. The 72h-NOEC for growth rate and yield inhibition was 100 mg/L.
Executive summary:

The objective of the study was to evaluate the test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield based on the OECD guideline No. 201.  

A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures per group were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition, three replicates per group were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L, in the combined range-finding test. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.  

Samples taken from the highest test concentration were analysed. The measured concentration was at 104-109% relative to the nominal concentration of 100 mg/L throughout the test. Based on these results, effect parameters were expressed in terms of the analytically confirmed nominal concentration.  

No inhibition of growth rate or yield was found at any test concentration. Contrary, algae exposed to the test item showed rather stimulation than reduction of growth. However, no significant differences were recorded between the values for growth rate or yield at the highest test concentration when compared to the control group. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 72h-EC50 for growth rate (ERC50) and yield (EYC50) inhibition was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L. The 72h-NOEC for growth rate and yield inhibition was 100 mg/L.

Description of key information

Under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of the test item. The 72h-EC50 for growth rate (ERC50) and yield (EYC50) inhibition was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L. The 72h-NOEC for growth rate and yield inhibition was 100 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The objective of the study was to evaluate the test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield based on the OECD guideline No. 201.  

A combined limit/range-finding test was performed. Six replicates of exponentially growing algal cultures per group were exposed to an untreated control and to a nominal concentration of 100 mg/L, in a limit test. In addition, three replicates per group were exposed to nominal concentrations of 0.10, 1.0 and 10 mg/L, in the combined range-finding test. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.  

Samples taken from the highest test concentration were analysed. The measured concentration was at 104-109% relative to the nominal concentration of 100 mg/L throughout the test. Based on these results, effect parameters were expressed in terms of the analytically confirmed nominal concentration.  

No inhibition of growth rate or yield was found at any test concentration. Contrary, algae exposed to the test item showed rather stimulation than reduction of growth. However, no significant differences were recorded between the values for growth rate or yield at the highest test concentration when compared to the control group. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 72h-EC50 for growth rate (ERC50) and yield (EYC50) inhibition was beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of 100 mg/L. The 72h-NOEC for growth rate and yield inhibition was 100 mg/L.