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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted at only one dose point and lacked experimental detail.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Details on test material:
As per IUCLID5 Sections 1.1. - 1.4.
Specific details on test material used for the study:
The VeoVa-9 test compound (sample No. 1141) was received from Shell Development Company on August 14, 1991 and assigned ABC Laboratories’ reference #TS—S 121. The compound was stored at room temperature and was observed to be a clear liquid. Compound purity was specified as 100%. This compound was used in the preparation of all preliminary and def‘initive test concentrations. This sample was also used for the preparation of analytical technical standards and primary stock solutions.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples taken at 0 and 96 hr.

Test solutions

Vehicle:
no
Details on test solutions:
The test solution was prepared in algal media such that the final nominal concentration was 18 mg/L. The stock solution was stirred for approximately one hour, and allowed to equilibrate for 96 hours before use.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The parent stock of Selenastrwn capricomutum Printz (Supplier Batch #1648, ABC Laboratories culture 91-26) used in the definitive test was obtained from The Department of Botany, Culture Collection of Algae, The University of Texas at Austin, Austin, Texas on November 21, 1991. The parent stock of algae was received growing on an agar slant contained in a 50-mL culture tube. The algae culture was identified as Selenastrum capricornutum Printz on the culture tube label. Scrapings were taken from the slant and placed in 250-mL flasks containing 100 mL of sterile algae nutrient media. These lots of algae were placed in an environmental chamber at 24 +/- 2 °C to allow the algae to grow. The parent culture was also divided into individual lots by adding single scrapings from the algae/agar surface to sterile culture tubes containing agar. The prepared lots were stored in an environmental chamber at 20 +/- 2 °C. Periodically new Selenastrum capricornutum Printz cultures were initiated using a lot of this parent stock or cloned from an existing culture derived from the parent stock in 100 mL of sterile culture medium. Cultures of Selenastrum capricornutum Printz at ABC Laboratories were maintained under the same environmental test conditions as for the definitive test. The algal culture used for this toxicity test was four days old at test initiation.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None

Test conditions

Hardness:
No data
Test temperature:
No data
pH:
No data
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
18 mg/L nominal, 12 mg/L measured
Details on test conditions:
Algae Nutrient Medium Preparation

The algal toxicity study was conducted in 250-mL Erlenmeyer flasks containing 100 mL of synthetic algae nutrient medium. This medium was composed of 1.0 mL of each of the following nutrient solutions diluted to a final volume of 1000 mL with autoclaved ABC reagent water.

Macronutrient Stock Solutions (each in 1000 mL)
NaNO3 25.500 g
NaHCO3 15.000 g
MgSO4°7H20 14.700 g
MgCl2°6H20 12.164 g
CaCl2-2H20 4.410 g
K2HP04 1.044 g



Micronutrients Stock Solution (in 1000mL)

MnCl2 4H20 415.4 mg
Na2EDTA 2H2O 300.0 mg
H3BO3 185.5 mg
FeCl3 6H20 159.8 m .
Na2MoO4 2H20 7.3 mg
ZnCl2 3.3 mg
CoCl2 6H20 1.4 mg
CuCl2 2H20 12.0 ug

After preparation, the medium was pH-adjusted to 7.5 +/- 0.1 (using 1 N NaOH) and re-sterilized by passage through Millipore® 0.45-MM filters. A total of eight liters were prepared for definitive testing.

Biological
The algal cell counts were accomplished utilizing a hemacytometer and an Olympus® Model BH-2 microscope. The hemacytometer has two chambers each with nine squares, 1 mm on a side. The average number of cells per 1 mm^2 was designated as "Q". The center square was subdivided into twenty—five 0.20-mm squares. The average number of cells per 0.04 mm2 was designated as "R". The cell density (d) for a given suspension of algal cells could be calculated from either of the following equations:

A. d(cells/mL) = 10^4 x Q (Average number of cells/1 mm^2)

B. d(cells/mL) = 10^6 X 1/4R (Average number of cells/0.04 mm^2)

In general, equation B was only used with very dense cell populations such as those encountered with a 4 to 7-day old algal culture typically used in the preparation of an algal test inoculum. Algal cell counts at 24, 48, and 72 hours of the definitive study used equation A and the 96 hour cell counts used equation B. When the average number of cells per l—mm square was less than 11 algal cells, all nine l-mm squares were counted and divided by nine to obtain the average number of cells per 1-mm square (Q) or the replicate mean value. For all suspensions with at least 11 cells per 1-mm square, the four corner 1—mm squares could be counted and averaged to obtain "Q" (replicate mean value). The cells/mL for each replicate was calculated and recorded.

Prior to the initiation of the definitive study, two 96-hour preliminary studies were conducted to determine the concentration range for the definitive study. The first preliminary study was conducted at the test levels of 1.0, 10 and 100 mg/L. The test solutions were prepared from diluted aliquots of the 100 mg/L test solution which had been magnetically stirred for approximately 42 hours. After 96 hours, algal cell counts for each of the test levels were 81, 80, and 72% of the control population, respectively.

Following the preliminary study, water solubility of VeoVa-9 was determined by ABC Laboratories, Inc. The solubility study titled, "Determination of the Water Solubility of VeoVa—9 by Generator Column Method" determined 13.1 +/- 0.86 mg/L to be the solubility of VeoVa-9 in water. The results of the solubility study will be presented to Shell Development Company as ABC Laboratories report #39579.

The solubility study results along with the first preliminary study results were used to determine the test concentration used for the second preliminary study. A nominal test concentration of 18 mg/L was used for the second preliminary study. The test solution was stirred magnetically for one hour and then remained undisturbed for approximately 96 hours. The preliminary test was initiated following the solution preparation procedure. Cell counts for the second preliminary study were made at 24, 48, 72, and 96 hours. At 0 hour, cell counts were preformed only on the control replicates, The 18 mg/L test concentration was determined to be 90% of the control population at 96 hours.

Based on the results of the solubility and preliminary studies, the nominal test concentration of 18 mg/L, including a control was selected for the definitive algal assay. All test flasks were labeled with a felt marker as to compound code, concentration, replicate and grid position. Each flask was stoppered with a foam plug.

Following preparation, the test vessels were positioned in random fashion and incubated for 96 hours at 24 +/- 2 °C under continuous cool-white fluorescent lighting and constant rotary agitation. Light intensity was maintained at 400 +/- 10% ft-c (approximately 4300 LUX) and the agitation rate was approximately 100 rpm. Temperature, light intensity and oscillation. rate were monitored throughout the study as shown in Table I. A continuous temperature recording of the test chamber was maintained for the duration of the study witl‘i‘a Rustrak® RangerTM Data Logger.

Chemical and Physical

For definitive testing, a 18 mg/L primary standard was prepared by weighing 0.0178 g of test material into a l-L volumetric flask and diluting with algae nutrient media. The 18 mg/L primary standard was magnetically stirred for one hour and then remained undisturbed for approximately 96 hours.

At 0 and 96 hours of the definitive study, measurements of temperature and pH were measured in the control and test solution. The test level and control were prepared in triplicate using 100 mL of the appropriate solution for each test vessel. Each test flask received 1.0 mL of algal inoculum containing approximately 1.0 x 10^6 cells/mL resulting in approximately 1.0 X 10^4 cells/mL for each flask. Initial cell counts of the control flasks resulted in an actual mean cell count of 9.3 x 10^3 cells/mL.
To determine the concentration of VeoVa-9 in a closed system, a sealed test solution was prepared at O-hour. Approximately 16.5 mL of the 18 mg/L test solution was placed in a glass test tube and then sealed with a teflon lined cap so that no head space was present. The sealed test solution was sampled at 96-hours.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
12 mg/L
Nominal / measured:
meas. (arithm. mean)
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 12 mg/L
Nominal / measured:
meas. (arithm. mean)
Basis for effect:
cell number
Details on results:
Prior to the initiation of the definitive study, a solubility study and two 96—hour preliminary. studies were conducted to determine the concentration range for the definitive study. The first preliminary study was conducted at the test levels of 1.0, 10 and 100 mg/L. After 96 hours, algal cell counts for each of the test levels were 81, 80 and 72 % of the control population. The second preliminary study was conducted at the test level of 18 mg/L. After 96 hours, the 18 mg/L test concentration was determined to be 90% of the control population. The solubility study determined that 13.1 +/- 0.86 mg/L was the solubility of VeoVa—9 in water.

A method validation was conducted to detemiine the recovery of VeoVa—9 in hard blended test water, prior to the initiation of the VeoVa-9 toxicity test. The results of the method validation were presented to Shell Development Company as ABC laboratories report #39571. Fortification concentrations for the method validation ranged from 0.250 mglL to 25.0 mg/L and yielded mean recoveries of 94 i 8.6% on an initial validation and 90 j: 14% on a second validation performed for purposes of verification. During the definitive study, the fortification concentration (Table II) was 18.0 mg/L and yielded an average recovery of 96 +/- 6.8%.

The results of the determination of VeoVa-9 in the test solutions during the Selenastrum capricornutum toxicity test are presented in Table III. The mean of the 0- and 96-hour measurements of VeoVa-9 is not reported due to the unquantifiable results at the termination of toxicity testing. The reason for this is based on the volatility of VeoVa-9 under algal test conditions in which the VeoVa-9 was in an unsealed test chamber exposed to warmth and constant shaking. The VeoVa—9 was almost completely volatilized from the samples at termination. Further proof of this can be found in the results of the sealed sample that was analyzed at 0 and 96 hours, along with the other samples. It provided an overall mean of 12 mg/L and showed only a minimal decrease in measured concentration at termination. The reported measured value of VeoVa-9 is based on the mean of the 0-hour results which was 12 mg/L. These results yield an average of 67% of nominal.

A 96 hour static acute algae study with VeoVa-9 was successfirlly completed on January 7, 1992. The nominal concentration of VeoVa—9 was selected for testing based on the results of the preliminary tests. Cell counts were conducted at 24, 48, 72, and 96 hours for the test concentration and control. Initial cell counts at 0 hour were performed only on the control replicates.

The growth data (cell counts) from the definitive test are presented in Table IV and Figure 1. The control and the 18 mg/L test level cell count data were subjected to a student t-test that indicated no significant difference (p<0.05) between them after 72 and 96 hours. Logarithmic phase growth was confirmed at 96 hours with a mean count of 3.3 X 10^6 cells/mL in the control replicates, which was a 355-fold increase from the initial 9.3 X 10^3 cells/mL.

The 24-, 48-, 72-, and 96-hour EC50 values for VeoVa-9 based on algal growth were estimated to be >12 mg/L, respectively. The 96-hour no observed effect level based on no growth inhibition, was determined to be 12 mg/L.

At 0 hour, temperature and pH were measured in the control and residual parent test solution. At 96 hours, temperature and pH were measured in all replicates of the control and test solution (Table V). At 0-hour, the temperature was 24°C and the pH ranged from 7.3 to 7.4. After 96 hours, the temperature was 25°C and the pH ranged from 7.6 to 7.8.

The definitive algal assay was performed according to ABC Laboratories protocol TSCA 797.1050 which conforms with the TSCA Guidelines. However, the nutrient solution used for culturing and testing of Selenasn'um capricomutum Printz contains the chelate EDTA which is recommended by ASTM (3). Studies performed by ABC Laboratories and others (3) have shown EDTA to be an essential nutrient to reach logarithmic phase growth and is necessary in the culturing of Selenastmm capricomutum Printz. The studies carried out by ABC Laboratories consisting of a control group containing EDTA vs. non-EDTA solution have conclusively indicated that the presence of EDTA was necessary for growth. Since organic chelators are a natural environmental constituent, the presence of EDTA in the nutrient media is both reasonable and necessary (3). For the preliminary and definitive studies, the nutrient solution contained 300 ug/L of EDTA which is necessary for algae growth.

The study was conducted following the Good Laboratory Practice regulations (4) and the final report was reviewed by ABC Laboratories’ Quality Assurance Unit. All original raw data were provided to Shell Development Company with the final report, and a copy was retained at ABC Laboratories, Inc.
Reported statistics and error estimates:
The SAS program (2) prints out the raw data, plots, charts, and computes the mean number of cells by treatment and hour, and tests for significant differences from the control and test level mean at each time point. A student t-test was conducted to determine if the control and test level
cell count values were significantly different (p50.05) from each other. The test indicated that there was not a significant difference after 72 and 96 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The nominal test concentration of VeoVa-9 tested for this study was 18 mg/L. The mean measured value was 12 mg/L based on 0-hour results. The 96 hour EC50 value for Selenastrum capricomutum Printz exposed to VeoVa-9 was >12 mg/L and the no observed effect level was 12 mg/L, based on the absence of a growth inhibitibn effect.
Executive summary:

The 96 -hour EC50 value for Selnastrum capricornutum exposed to vinyl neononanoate under static conditions was > 12 mg/L and the no observed effect level was 12 mg/L, based on the absence of a growth inhibition.