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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:
March - August, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
The non-GLP certificate of analysis lists the test material as containing 0.18% ethylene glycol and having a viscosity of 33633 cPs at 25°C.
The record of custody lists the test material as 100% of the desired substance.
The record of custody lists the test material as having a 2 year shelf life.
Analytical monitoring:
yes
Details on sampling:
Samples were collected from the bulk test solutions at test initiation and from pooled replicate samples at exposure termination. Blank replicates were analyzed separately. All test solutions were analyzed within 24 hours of
preparation; therefore, no stability assessment was required.
Vehicle:
no
Details on test solutions:
Results of non-GLP preliminary methodology work indicated that the test material was not expected to be completely soluble at the selected test concentrations for the study. Analytical results of a preliminary non-GLP trial indicated that a dosing (loading rate) of 100 mg EPON™ Resin CS-337/L required approximately seven days of slow stirring to reach a equilibrated saturated state (i.e., ≤ 20% measured concentrations between successive sampling events). Based on this information, the study used the water accommodated fraction (WAF) preparation method to prepare the test solutions. Following direct addition of the test material into AAP medium and the appropriate mixing time (seven days), the clear portion (i.e., no visible undissolved test material) of the highest loading rate (10 mg/L) was siphoned to produce a WAF test solution. The 10 mg/L bulk WAF solution was clear and colorless. Due to limitations of weighing small amounts of the test material, subsequent loading rates (test solutions) were prepared as serial dilutions of the 10 mg/L WAF test solution.The medium control consisted of AAP medium without the addition of the test material. The prepared bulk WAF test solutions were apportioned into individual test vessels. Remaining bulk solutions were clear and colorless, including the AAP medium control. The test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solution was not required. The dispersal of the test material in the surrounding medium (AAP) was considered to represent the most probable route of exposure in the environment.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata from in-house cultures was initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 060215) in June 2015. This species is widely accepted and recommended for toxicity testing by the test
guidelines. Stock cultures of this organism were maintained axenically by periodic transfer into sterile medium. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2ºC. The algal inoculum for the test was prepared from a 4-day old stock culture. A Coulter Multisizer 3 (Beckman Coulter, Brea, California) was used to determine the cell density of the stock culture. This evaluation determined that a 0.075 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 10,000 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
Temperature during the exposure period ranged from 22-23 ºC.
pH:
The pH of the bulk solutions (no algae) were 7.0 at test initiation. At test termination the pH ranged from 7.4 to 7.5 and 7.0 to 7.1 in replicates with and without algae, respectively.
Nominal and measured concentrations:
nominal loading rates: 0 (AAP control), 0.030, 0.10, 0.30, 1.0, 3.1 and 10 mg EPON™ Resin CS-337/L.
Details on test conditions:
The algae was cultured in freshwater algal nutrient medium (i.e., AAP medium), prepared with sterile deionized water and reagent grade chemicals. The water source for the
deionized water system was Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant prior to treatment. The base water used to prepare the medium was passed through a series of activated carbon, (two) deionization polymer (US Filter Mixed Bed, Type 1), and a final filtration unit, prior to collection and autoclaving in clean glass containers. Prior to treatment, the base water used to prepare the medium is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results.

The definitive test was conducted under static conditions for approximately 72 hours from 28 to 31 March 2017. Three replicate test vessels were prepared per test level. Six replicates were prepared for the AAP medium control. Test vessels were sterilized 250-mL borosilicate Erlenmeyer flasks with foam stoppers. Each flask was uniquely labeled (i.e., study, replicate, test level) for identification purposes. Each replicate test vessel contained 50 mL of the appropriate test solution and was inoculated with approximately 10,000 cells/mL. An additional replicate at each test level and control was prepared but not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the potential interference of the test material and to monitor pH without the algal biomass. At test initiation and following sampling for cell densities at 24 and 48 hours, the replicate test vessels were placed in a walk-in environmental chamber (Lab-Line Environmental Chamber, Lab-Line Inc., Melrose, Illinois) on a shaker table (set at approximately 100 rpm) according to a computer-generated randomization. The target test temperature was 23 ± 2ºC. The photoperiod was set at 24 hours of continuous light with a target light intensity of 5,200 ± 780 lux. The pH was measured from bulk test solutions at 0 hours and from blanks and pooled replicate samples of each test level and AAP medium control at 72 hours. Pooled samples were prepared by withdrawing and combining approximately 2.5-mL volumes from each inoculated replicate at each test level and AAP medium control. Temperature was continuously monitored with a minimum/maximum thermometer placed in a representative vessel which was incubated adjacent to the test material exposures. At test initiation, light intensity was measured at each position where inoculated replicate vessels were placed during the exposure. Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea,California) fitted with a 100-μm aperture tube. Total cell counts were determined at approximately 24, 48 and 72 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. Two cell count readings were made per replicate and averaged. The readings for the blank replicates were used to correct for background in daily calculations. In addition, morphological observations were done at test termination on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus BH Microscope (Olympus Corporation, Tokyo, Japan);20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck).
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
4.65 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
9.96 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
Measured concentrations of the major structural component of EPON™ Resin CS-337 in the 0.10, 0.30, 1.0, 3.1 and 10 mg/L bulk test solutions at test initiation were 0.00614, 0.0114, 0.0426, 0.161 and 1.69 mg/L, respectively. Measured concentrations of the major structural component of EPON™ Resin CS-337 in pooled replicate test solutions (with algae) at exposure termination were 0.00672, 0.0186, 0.0499, 0.189 and 1.81 mg/L, equating to 109, 163, 117, 117 and 107% of the measured concentrations at test initiation, respectively. Measured concentrations of the major structural component of EPON™ Resin CS-337 in blank replicate test solutions (without algae) at exposure termination were 0.00436, 0.0118, 0.0285, 0.120 and 1.49 mg/L, equating to 71.0, 104, 66.9, 74.5, and 88.2% of the measured concentrations at test initiation, respectively. None of the analyses of the controls or the 0.030 mg/L loading rate exhibited an EPON™ Resin CS-337 concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.0025 mg EPON™ Resin CS-337/L (major structural component). Measured concentrations of each loading rate were used to verify test solution preparations and associated stability over the course of the exposure period. The endpoints are reported using the nominal loading rates. These results indicate that the presence of algae had no effect on test material exposure concentrations in the test vessels.

Mean yields at 72 hours were 265.8, 222.9, 286.8, 298.8, 291.1, 230.9 and 156.4 (x104) cells/mL for the AAP medium control, 0.030, 0.10, 0.30, 1.0, 3.1 and 10 mg EPON™ Resin CS-337/L test levels, respectively. At 72 hours, the mean inhibition response relative to the control ranged from -12 to 41% inhibition of yield. At 72 hours, cell yield at the 10 mg EPON™ Resin CS-337/L test level was significantly different from the AAP medium control. Thus, the 72-hour NOELR for yield is reported as 3.1 mg EPON™ Resin CS-337/L.
The calculated EyL50 is >10 mg/L (highest loading rate tested). The calculated EyL20 (95% confidence intervals) is 4.65 (2.29 – 9.45) mg EPON™ Resin CS-337/L and the calculated EyL10 (95% confidence intervals) is 2.80 (0.919 – 8.54) mg EPON™ Resin CS-337/L.

Mean specific growth rates between 0 and 72 hours were 1.861, 1.802, 1.886, 1.900, 1.892, 1.809 and 1.681 (day-1) for the AAP medium control, 0.030, 0.10, 0.30, 1.0, 3.1 and 10 mg EPON™ Resin CS-337/L test levels, respectively. From 0 to 72 hours, mean inhibition response relative to the control ranged from -2 to 10% of the mean specific growth rate. At 72 hours, mean specific growth rate at the 10 mg EPON™ Resin CS-337/L test level was significantly different from the AAP medium control. Thus, the 72-hour NOELR for growth rate is reported as 3.1 mg EPON™ Resin CS-337/L. The calculated ErL50 and ErL20 are >10 mg/L (highest loading rate tested). The calculated ErL10 (95% confidence intervals) is 9.96 (7.17 – 13.8) mg EPON™ Resin CS-337/L.

At test termination, microscopic evaluation of algal cells at each loading rate,including the AAP medium control, revealed no abnormal observations.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity values for Pseudokirchneriella subcapitata exposed to EPON™ Resin CS-337 over a 72-hour static exposure period and nominal loading rates were as follows:
o Cell yield
72-hour EyL50: >10 mg/L (highest loading rate tested)
72-hour EyL20: 4.65 mg/L (95% CI = 2.29 – 9.45 mg/L)
72-hour EyL10: 2.80 mg/L (95% CI = 0.919 – 8.54 mg/L)
72-hour NOELR: 3.1 mg/L
o Growth rate
72-hour ErL50: >10 mg/L (highest loading rate tested)
72-hour ErL20: >10 mg/L (highest loading rate tested)
72-hour ErL10: 9.96 mg/L (95% CI = 7.17 – 13.8 mg/L)
72-hour NOELR: 3.1 mg/L
Executive summary:

The purpose of this study was to assess the potential effects of EPON™ Resin CS-337 to the freshwater green alga, Pseudokirchneriella subcapitata. Due to the limited solubility of this multicomponent test material, the study used the water-accommodated fraction (WAF) preparation method to prepare the test solutions. The study was performed for 72 hours with target nominal loading rates of 0 (algal assay procedure medium (AAP)), 0.030, 0.10, 0.30, 1.0, 3.1 and 10 mg EPON™ Resin CS-337/L. Cell density was determined at approximately 24, 48 and 72 hours (±1 hour from exposure initiation) Temperatures during the exposure period ranged from 22-23°C. The pH ranged from 7.0-7.5 and the light intensity ranged from 4550-5660 lux. Test solutions were analyzed for the major structural component of EPON™ Resin CS-337 at test initiation and exposure termination by high performance liquid chromatography mass spectrometry (HPLC/MS-MS). None of the analyses of the AAP medium control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.0025 mg EPON™ Resin CS-337/L (major structural component). Measured concentrations of the major structural component of EPON™ Resin CS-337 at exposure termination ranged from 66.9 to 163% of the measured concentrations at test initiation. The measured concentrations of the test solutions were used to verify test solution preparations and associated stability over the course of the exposure period. Study endpoints are reported using the nominal loading rates. The acute toxicity values for Pseudokirchneriella subcapitata exposed to EPON™ Resin CS-337 over a 72-hour exposure period and based on nominal loading rates were as follows:

o Cell yield

72-hour EyL50: >10 mg/L (highest loading rate tested)

72-hour EyL20: 4.65 mg/L (95% CI = 2.29 – 9.45 mg/L)

72-hour EyL10: 2.80 mg/L (95% CI = 0.919 – 8.54 mg/L)

72-hour NOELR: 3.1 mg/L

o Growth rate

72-hour ErL50: >10 mg/L (highest loading rate tested)

72-hour ErL20: >10 mg/L (highest loading rate tested)

72-hour ErL10: 9.96 mg/L (95% CI = 7.17 – 13.8 mg/L)

72-hour NOELR: 3.1 mg/L

Description of key information

The acute toxicity values for Pseudokirchneriella subcapitata exposed to EPON™ Resin CS-337 over a 72-hour exposure period and based on nominal loading rates were as follows:

72-hour EL50 (growth rate): >10 mg/L (highest loading rate tested)

72-hour EL20 (growth rate): >10 mg/L (highest loading rate tested)

72-hour EL10 (growth rate): 9.96 mg/L (95% CI = 7.17 – 13.8 mg/L)

72-hour NOELR (growth rate): 3.1 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
10 mg/L
EC10 or NOEC for freshwater algae:
3.1 mg/L

Additional information