Avermectin A1a, 25-cyclohexyl-5-O-demethyl-25-de(1-methylpropyl)-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 Feb to 07 Mar 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP.

Qualifier:

according to guideline

Guideline:

other: Environmental Assessment Technical Assistance Document 4.01, Algal Assay (U.S. Food and Drug Administration, 1987)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At test initiation, a single sample was removed from the stock solution used to prepare the test solutions and from the flasks containing the test solutions for analysis of test substance concentration. At test termination, a single composite sample from the three replicate vessels of each treatment and control group was analyzed for test substance concentration.

Vehicle:

yes

Details on test solutions:

A 0.0432 g portion of test substance was weighed into a sterile beaker, rinsed into a 100 mL volumetric flask with acetone and diluted to volume with acetone to create a stock solution of 403.5 μg A.I./mL. Appropriate volumes of this stock solution were transferred to 500 mL volumetric flasks and diluted to volume with sterile MBL medium to create nominal test solution concentrations of 40 and 20 μg A.I./L.
A 500 mL control solution was also prepared containing algal growth medium only. A solvent control solution was prepared containing 0.05 mL acetone diluted to 500 mL with algal growth medium. The concentration of acetone in the solvent control (100 μg/L) was equivalent to the amount of acetone present in each test solution.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green alga
- Strain: Selenastrum capricornutum
- Source (laboratory, culture collection): Carolina Biological Supply Company, Burlington, North Carolina
- Age of inoculum (at test initiation): 2-day old
- Method of cultivation: The culture medium used was Marine Biological Laboratory (MBL) medium prepared with distilled deionized water and adjusted to pH 7.5 ± 0.1 with 0.1 N hydrochloric acid after autoclaving. Stock cultures were grown in 125-mL glass flasks containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The stock cultures were maintained under test conditions (shaking rate of 100 rpm, temperature of 22-24℃, continuous illumination at the surface of the medium of approximately 5,000 lux) (SLI Algae Daily Log, 1990). Temperature was controlled using an environmental chamber. Lighting was supplied by Cool-White® and Vita-Lite® fluorescent lights. Culture flasks were agitated continuously on an orbital shaker.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None stated

Hardness:

None stated

Test temperature:

22-24℃

pH:

7.5±0.1

Dissolved oxygen:

None stated

Salinity:

None stated

Nominal and measured concentrations:

Nominal concentrations: 40 and 20 μg A.I./L
Measured concentrations: 26 and 14 μg A.I./L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 125-mL flasks
- Material, size, headspace, fill volume: Fifty-mL aliquots of the stock solution were removed and transferred to the test vessels.
- Aeration: All test vessels were fitted with stainless steel caps to permit gas exchange.
- Initial cells density: 1E+04 cells/mL
- No. of organisms per vessel: 6500
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
The culture medium used was Marine Biological Laboratory (MBL) medium prepared with distilled deionized water and adjusted to pH 7.5 ± 0.1 with 0.1 N hydrochloric acid after autoclaving.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algal growth medium (MBL medium minus Na2EDTA.2H2O) prepared with distilled deionized water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: adjust to 7.5 ± 0.1 with 0.1 N hydrochloric acid
- Light intensity: Lighting was provided continuously at an intensity of 4,000-5,000 lux at the solution surface by a combination of Cool-White® and Vitalite® fluorescent bulbs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At each 24-hour interval cell counts were conducted on each replicate vessel using a hemacytometer (distributed by American Optical Corporation) and Bausch&Lomb compound microscope.
Water quality (pH and conductivity) was measured at test initiation and termination.
Temperature was measured continuously with a Brooklyn Thermometer Company minimum/maximum thermometer. The shaking rate of the orbit shakers was recorded daily. The light intensity of the test area was measured with a General Electric type 214 light meter at 0 hour and each 24-hour interval of the exposure period.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: nominal concentrations of 1.0, 0.10, 0.010 and 0.0010 mg A.I./L
- Results used to determine the conditions for the definitive study: The preliminary exposure indicated that test substance was not acutely toxic to Selenastrum capricornutum. Therefore, a definitive study was conducted at two concentrations of 20 and 40 μg A.I./L.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: cell density and growth rate

Details on results:

- Cell density:
Cell densities were observed to increase over time in all replicates of each treatment level, the control and solvent control. Because of cell clumping, beginning at day 10, a 1 mL subsample was removed and sonicated for two minutes and then counted. The subsample was discarded after counting. Throughout the test, cells were observed to be normal in all treatment and control cultures. Results of the t-test demonstrated a significant difference between the maximum cell density of the control cultures and that of the solvent control cultures. Consequently, the treatment data were compared to the solvent control data using a two-tailed Dunnett's Test. The maximum cell densities observed at 14 and 26 μg A.I./L were determined to be statistically similar to the solvent control data.
- Growth rate:
Results of the t-test demonstrated no significant difference between growth rates of the control cultures as compared to the solvent control cultures, therefore, data from the two sets of controls were pooled. Bonferroni's Test demonstrated that the maximum growth rates for the 14 and 26 μg A.I./L treatment levels were statistically similar (p≤0.050) to the pooled control data.
- Causes of difference between measured and nominal values:
Possible causes in the decline of test substance concentration include uptake or sorption by algae, removal by physical processes (e.g., photolysis or hydrolysis) and/or biological degradation. This apparent decrease in test material concentrations can be attributed to the propensity of the test substance to adhere to glass surfaces as well as absorb to particulate matter inherent in the test system. In addition, the test substance is also photolytically unstable with a half-life of 12.2 hours in water under artificial sunlight conditions.

Reported statistics and error estimates:

A t-test was conducted to statistically compare the maximum cell densities and the maximum growth rates for the two control data sets. If control and solvent control data were similar (p≤0.05), the two control sets were pooled for further analyses. If a signficant difference was found between controls and solvent controls, data from the solvent control was used for statistical comparison.
From the observed values for maximum culture density and the calculated values for maximum growth rate, the highest test concentration that caused no statistical adverse effect (No Observed Effect Limit, NOEL) was determined using a two-tailed Dunnett's Test (Dunnett, 1955, 1964) when the treatment data were compared to the solvent control or using Bonferroni's Test (Weber, et al, 1989) when the control sets were pooled. The data were first checked for normality using the Shapiro-Wilks Test (Weber et al,1989) and for homogeneity of variance using Bartlett's test or Bonferroni's Test. The lowest test concentration that caused a significant effect (Minimum Inhibitory Concentration, MIC) was also determined from these analyses.

Validity criteria fulfilled:

yes

Conclusions:

For maximum cell density and maximum growth rate, the No Observed Effect Limit (NOEL)=26 μg A.I./L (0.026 mg/L) and the Minimum Inhibitory Concentration (MIC) was estimated to be >26 μg A.I./L (>0.026 mg/L), the highest concentration tested. This concentration is approximately the maximum solubility of the test substance in water.

Description of key information

One algae study is available. For maximum cell density and maximum growth rate, the No Observed Effect Limit (NOEL)=0.026 mg/L and the Minimum Inhibitory Concentration (MIC) was estimated to be >0.026 mg/L, the highest concentration tested (Giddings, 1992).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.026 mg/L

Additional information

One algae study was conducted according to FDA Technical Assistance Document 4.01 under GLP (Giddings, 1992).

For maximum cell density and maximum growth rate, the No Observed Effect Limit (NOEL)=26 μg A.I./L (0.026 mg/L) and the Minimum Inhibitory Concentration (MIC) was estimated to be >26 μg A.I./L (>0.026 mg/L), the highest concentration tested. This concentration is approximately the maximum solubility of the test substance in water.