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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:
October 08 to 11, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) and EU method C.3 (Algal Inhibition Test). Study was GLP certified.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Test solution samples were collected on Day 0 from the newly prepared batches of test solution to determine concentrations of the test substance. At test termination, composite samples were collected from each of the three remaining replicates from each treatment and control group for analysis of test substance concentrations. All samples were placed in glass vials and processed immediately for analysis.
Vehicle:
no
Details on test solutions:
Individual test solutions were prepared at nominal concentrations of 6.3, 13, 25, 50 and 100 mg a.i./L by mixing calculated volumes of diethylhydroxylamine into freshwater algal medium in approximately 4.0-L glass aspirator bottles. The bottles were completely filled to minimize headspace, and were stoppered during mixing to minimize volatilization. The solutions were stirred on magnetic stir plates for approximately 20 minutes, and appeared clear and colorless after mixing.
Each solution was continuously stirred while it was decanted into the test chambers through a spigot and tubing approximately two to three cm from the bottom of the aspirator bottle. Each test chamber was filled completely to minimize headspace and was capped with a glass stopper. No precipitates were observed in any of the test solutions at test initiation or termination.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater green alga, Pseudokirchneriella subcapitata Printz (UTCC 37) were obtained from the University of Toronto Culture Collection, and had been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland. Algal cells used in this test were obtained from Wildlife International, Ltd. cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. The culture was last transferred to fresh medium three days prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 72-hour exposure period. Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
23.9-24.5°C
pH:
From 7.8 to 8.0 on Day 0, and from 10.2 to 10.5 by Day 3.
The pH tended to increase relative to algal population, which is typical for tests conducted with Pseudokirchneriella subcapitata.
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations tested were 6.3, 13, 25, 50 and 100 mg a.i./L. Measured concentrations on Day 0 ranged from approximately 95 to 104% of nominal concentrations. Measured concentrations on Day 3 ranged from approximately 50 to 88% of nominal concentrations, and recoveries were generally lower in those treatment groups that exhibited the highest algal growth. Since the recovery on Day 3 in the 100 mg a.i./L solution was within 20% of the initial concentration, the material appears to be stable over the 72-hour exposure period. In addition, during the course of the test, the decrease in measured concentration in the lower concentration solutions was not accompanied by a decline in growth inhibition, suggesting that the material was not lost from the test system, but was likely due to adsorption to the algal biomass. Therefore, the results of the study were based on the Day 0 measured concentrations of 6.1, 12, 26, 48 and 101 mg a.i./L.
Details on test conditions:
At test initiation an inoculum of algal cells was added to each test chamber to achieve a nominal concentration of approximately 5,000 Pseudokirchneriella cells/mL. Due to the volatile nature of the test substance, closed-bottle test chambers with minimal headspace were used. Nine replicate test chambers were tested in the negative control and each treatment group. Samples were collected from three replicate test chambers sacrificed at each approximately 24-hour interval during the test to determine cell densities (i.e., replicates A, B and C at 24 hours; replicates D, E and F at 48 hours; and replicates G, H and I at 72 hours). Cell densities were subsequently used to calculate areas under the growth curve (biomass), growth rates and yield. Cell densities, biomass, growth rates and yields were used to calculate percent inhibition values relative to the negative control over the 72-hour exposure period. EC50, EbC50 and ErC50 values (based on the theoretical test concentration that would produce a 50% reduction in cell density, biomass and growth rate, respectively) were determined at 24, 48 and 72 hours. An EyC50 value (based on the theoretical test concentration that would produce a 50% reduction in yield) was determined at 72 hours. A no-observed-effect concentration (NOEC) was determined at 72 hours through statistical evaluation of the cell densities, biomass, growth rates and yield, as well as examination of the concentration-response pattern.

Culture Medium:
The algal cells were cultured and tested in freshwater algal medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Wildlife International, Ltd. well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water (Appendix 3). The medium also contained supplemental sodium bicarbonate (35 mg/L). The total concentration of sodium bicarbonate in the medium was 50 mg/L. The pH of the medium was adjusted to 7.5 ± 0.1 using 10% HCl and 0.1 N NaOH, as needed, and the medium was sterilized by filtration (0.22 µm) prior to use.
Test Apparatus:
Test chambers were sterile, 300-mL glass BOD bottles with glass stoppers and were completely filled with medium to minimize head-space. Each test chamber contained two glass marbles to facilitate mixing. The test chambers were indiscriminately positioned daily on mechanical shakers in an environmental chamber designed to maintain the desired test temperature. The test flasks were shaken continuously at approximately 100 rpm. Each test chamber was labeled with the project number, test concentration and replicate.
Environmental Conditions:
Test chambers were held in an environmental chamber at a temperature of 23 ± 2°C. The temperature of a container of water adjacent to the test chambers in the environmental chamber was recorded twice daily during the test using a calibrated hand-held, liquid-in-glass thermometer. The algae were held under 24 hours per day of cool-white fluorescent lighting at an intensity of 6000 lux ± 20%. At test initiation, light intensity was measured at test solution level at five locations surrounding the test flasks on each mechanical shaker, using a SPER Scientific Model 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and at approximately 24-hour intervals thereafter using a Thermo Orion Model 720 Aplus pH meter. Samples for pH measurement at test initiation were collected from the individual batches of test solution prepared
for each treatment and control group. At 24, 48 and 72 hours, pH was measured in pooled samples of test solution collected from each of the three replicates sacrificed from each treatment and control group.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
26 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 101 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
26 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
Changes in mean cell density in the negative control replicates over the 72-hour exposure period indicated that exponential growth of cells occurred in those replicates (Figure 1). After 72 hours of exposure, percent inhibition of cell density in the 6.1, 12, 26, 48 and 101 mg a.i./L treatment groups was –6.7, –17, 1.7, 20 and 43%, respectively, relative to the negative control. Dunnett’s test indicated that cell density was significantly reduced in the 48 and 101 mg a.i./L treatment groups (p = 0.05). Therefore, the 72-hour NOEC for cell density was 26 mg a.i./L. Since there was <50% inhibition of cell density in all treatment groups, the 72-hour EC50 value for cell density was >101 mg a.i./L, the highest concentration tested.
After 72 hours of exposure, percent inhibition of biomass in the 6.1, 12, 26, 48 and 101 mg a.i./L treatment groups was –3.8, –17, –0.46, 14 and 37%, respectively, relative to the negative control. Dunnett’s test indicated that biomass was significantly reduced in the 48 and 101 mg a.i./L treatment groups (p = 0.05). Therefore, the 72-hour NOEC for biomass was 26 mg a.i./L. Since there was <50% inhibition of biomass in all treatment groups, the 72-hour EbC50 value for biomass was >101 mg a.i./L, the highest concentration tested.
After 72 hours of exposure, percent inhibition of growth rate in the 6.1, 12, 26, 48 and 101 mg a.i./L treatment groups was –1.4, –3.4, 0.40, 4.7 and 12%, respectively, relative to the negative control. Dunnett’s test indicated that growth rate was significantly reduced in the 48 and 101 mg a.i./L
treatment groups (p = 0.05). Therefore, the 72-hour NOEC for growth rate was 26 mg a.i./L. Since there was <50% inhibition of growth rate in all treatment groups, the 72-hour ErC50 value for growth rate was >101 mg a.i./L, the highest concentration tested.
After 72 hours of exposure, there were no noticeable changes in cell morphology at any of the concentrations tested when compared to the negative control. There were no signs of aggregation or flocculation of algae in the control or in any treatment group. Adherence of cells to the test chambers was evident in all treatment groups. However, it was comparable to the adherence noted in the negative control and was not considered to be related to treatment with the test substance.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Cell densities, biomass, growth rates and yields were analyzed statistically by non-linear regression versus concentration to estimate EC50 values (i.e., the theoretical test concentration that would produce a 50% reduction in cell density (EC50), biomass (EbC50), growth rate (ErC50) and yield (EyC50)) and the corresponding 95% confidence limits (5). The data were evaluated for normality and homogeneity of variance (p = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. The treatment groups then were compared to the control using Dunnett’s test (p = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC at 72 hours.
Validity criteria fulfilled:
yes
Remarks:
see conclusions
Conclusions:
Cell density increased in the negative control by a factor of at least 16 within three days. The coefficient of variation of average specific growth rates during the whole test period (0 - 72 hours) in the control replicates did not exceed 7%.
Executive summary:

The acute toxicity of N,N-Diethylhydroxylamine (DEHA) was evaluated with the freshwater green alga, Pseudokirchneriella subcapitata) in a study performed in accordance with OECD testing guideline 201 and GLP requirements. Due to the volatile nature of the test substance, closed-bottle test chambers with minimal headspace were used. The validity criteria were fulfilled. Measurements of pH ranged from 7.8 to 8.0 on Day 0, and from 10.2 to 10.5 by Day 3. The method used for the analysis of DEHA in algal medium samples was Method 8140, Iron Reduction Method for Oxygen Scavengers (Hach Company). It is not mentioned that the Hach test is able to discriminate between DEHA and its degradation product (the nitrone).

Day 0 measured concentrations of DEHA ranged from 6.1 to 101 mg a.i./L. The 72-hour EC50, EbC50, ErC50 and EyC50 values for cell density, area under the growth curve (biomass), growth rate and yield, respectively, were >101 mg a.i./L, the highest concentration tested. The 72-hour NOEC, based on cell density, biomass, growth rate and yield, was 26 mg a.i./L.

As the three validity criteria of the OECD guideline 201 were fulfilled, this study is considered as reliable without restriction.

Description of key information

 The acute toxicity of N,N-Diethylhydroxylamine was evaluated with the freshwater green algae, Pseudokirchneriella subcapitata, in a study performed in accordance with OECD testing guideline 201 and GLP requirements. Due to the volatile nature of the test substance, closed-bottle test chambers with minimal headspace were used. The validity criteria were fulfilled.
    
    

Measurements of pH ranged from 7.8 to 8.0 on Day 0, and from 10.2 to 10.5 by Day 3.The method used for the analysis of DEHA in algal medium samples wasMethod 8140, Iron Reduction Method for Oxygen Scavengers (Hach Company). It is not mentioned that the Hach test is able to discriminate between DEHA and its degradation product (the nitrone).

The 72-hour EC50, EbC50, ErC50 and EyC50 values for cell density, area under the growth curve (biomass), growth rate and yield, respectively, were >101 mg a.i./L of DEHA, the highest concentration tested (initial measured concentration). The 72-hour NOEC, based on cell density, biomass, growth rate and yield, was 26 mg a.i./L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
26 mg/L

Additional information