2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 December 2006 to 22 December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

SAMPLE COLLECTION/EXTRACTION
Bulk dose solutions, pooled and blank spent test solutions (control algal assay medium (AAM), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg test material/L) were sampled for analytical confirmation on days 0 and 5 of the study. For the bulk dose and blank spent test solutions, a 2-mL aliquot was withdrawn from each test solution and stored at -80 °C. For the control pooled spent test solutions, equal volume aliquots from six replicate test vessels were combined to form a 2-mL aliquot, and then stored at -80 °C. For the remainder of the pooled spent test solutions, equal volume aliquots from triplicate test vessels were combined to form a 2-mL aliquot at each dose level, and then stored at -80 °C. Sample aliquots were thawed and analysed without dilution by liquid chromatography with ultraviolet detection (LC/UV).

METHOD PRECISION AND HOMOGENEITY
To assess analytical method precision and solution homogeneity, three additional samples were collected on day 0 from the 3.13 and 100 mg test material/L bulk dose solutions. These additional samples were collected and analysed along with the other day 0 samples as described above.

Vehicle:

no

Details on test solutions:

Bulk test solutions were prepared at test initiation (day 0) at nominal (target) concentrations of 0 (medium control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg test material/L. The target weight of test material added into a 1000-mL volumetric flask to achieve 100 mg test material/L, adjusted for purity was 105.982 mg (actual weight 105.92 mg). The test material was weighed out directly into a tared scintillation vial. Some AAM was then added to the vial (approximately half full), the top was placed on the vial, the vial was shaken, and then the solution was transferred to the volumetric flask. The vial was then rinsed several times with AAM using the same procedure and these rinses were added to the flask. Additional AAM was added to the volumetric flask (not into the neck of the flask), a stopper was placed on the flask, and the solution was shaken vigorously to mix thoroughly. The flask was then brought to volume with AAM, the stopper was replaced, and the solution was shaken vigorously again to mix. The stock solution was noted with some test material particles and was sonicated for approximately 3 minutes. Following sonication the test solution appeared slightly yellow but clear. This test solution also served as the stock solution for preparing the remaining test solutions.
The remaining test solutions were prepared as dilutions from the high-test solution. After transfer of stock solution, the test solutions were prepared in a similar fashion to the high concentration test solution. All test solutions were noted as clear. Test solutions were shaken vigorously prior to pouring off into test vessels and prior to analytical sampling. The medium control solution was the AAM.

Test organisms (species):

Anabaena flos-aquae

Details on test organisms:

TEST ORGANISM
- Strain: UTCC 67
- Source. Algae were from in-house cultures.
Stock cultures of this organism were maintained aseptically by periodic transfer into sterile medium.

CULTURING CONDITIONS
Temperature: 23 ± 2 °C
Light (lux): 2200 ± 440 (test subculture)
Photoperiod: Continuous (24 hours of light/day)
Medium: Algal assay medium (AAM) designated for the EPA algal assay bottle test
pH: Range: approximately 7.0–7.5
Culture Conditions: Axenic
Culture Volume: 100 mL
Culture Vessel: 500-mL Erlenmeyer flask
Culture Vessel Cap: Shimadzu closure

ALGAL INOCULUM
The algal inoculum for the test was prepared from a 3-day old stock culture. The target initial cell density was 20 000 cells/mL. The initial target cell density was set higher than normal to aid in reduction of a potential lag in growth during the early part of the exposure which can be an issue with Anabaena flos-aquae. A Bright-Line Haemocytometer was used to determine the density of the stock culture. This evaluation determined that a 3.8 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 20 000 cells/mL.
In addition, a Coulter Multisizer 3 was used to determine the cell volume of the stock culture. This would be used to calculate a nominal initial cell volume for use in calculation of growth rate based on cell volume since this would be the primary endpoint used to evaluate growth in the test vessels.
Since the volume of inoculum added (3.8 mL) was approximately 5 % of the volume of medium in each replicate (75 mL), the nominal initial cell density and cell volume were corrected to adjust the nominal values for the total volume in each replicate including the inoculum volume (78.8 mL).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

23.5- 23.7 °C

pH:

The pH values ranged from 7.3 to 3.3 at test initiation, from 7.6 to 3.5 in pooled replicates with algae on day 3, and from 9.3 to 3.5 in pooled replicates with algae and 7.3 to 3.5 in blank replicates without algae at test termination.

Nominal and measured concentrations:

0 (medium control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L (nominal)
< LLQ (medium control), 3.10, 5.62, 11.6, 23.3, 47.2 and 94.7 mg/L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 250-mL borosilicate Erlenmeyer flasks with foam stoppers each containing 75 mL test medium.
- Aeration: vessels were placed on a shaker table set at 100 rpm during the exposure period
- Initial cell density: 20 000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7
Four replicate test vessels were prepared per test level and seven replicate test vessels were prepared at the medium control level. Each replicate contained 75 mL of the appropriate test solution. Three replicates at each test level and six replicates at the medium control level were inoculated with approximately 20 000 cells/mL. Inoculations were made within 40 minutes of sampling for analytical verification of test solution concentrations. The additional replicate at each test and control level was not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass.

GROWTH MEDIUM
- Standard medium used: Yes. The growth and test medium used was that designed for the EPA Algal Assay Bottle Test (Miller et al., 1978). The medium was prepared using deionised water. The source water for the deionised water was Lake Huron water supplied to the test laboratory by the City of Midland Water Treatment Plant prior to treatment.
- Detailed composition: The composition of the Algal Assay Medium (AAM) was as follows: NaNO₃ (25.5 mg/L); MgCl₂.6H₂O (12.2 mg/L); CaCl₂.2H₂O (4.4 mg/L); MgSO₄.7H₂O (14.7 mg/L); NaHCO₃ (15.0 mg/L); K₂HPO₄ (1.044 mg/L); H₃BO₃ (0.186 mg/L); MnCl₂.4H₂O (0.417 mg/L); ZnCl₂ (0.00327 mg/L); Na₂MoO₄.2H₂O (0.00726 mg/L); CoCl₂.6H₂O (0.00143 mg/L); CuCl₂.2H₂O (0.000011 mg/L); Na₂EDTA.2H₂O (0.3 mg/L); FeCl₃.6H₂O (0.16 mg/L).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes. The pH of medium to be used on test was adjusted to 7.5 ± 0.1 prior to solution preparation
- Photoperiod: continuous light
- Light intensity and quality: 2050 - 2400 lux

EFFECT PARAMETERS MEASURED
At approximately 24, 48, 72, 96, and 120 hours, two samples were withdrawn from each replicate (1 mL per sample on days 1 and 2 and 0.5 mL per sample each on days 3, 4, and 5). The replicates samples were transferred to Isoton II diluent solution at the appropriate dilutions for determination of particle counts and cell volume on the Coulter Multisizer 3. Each replicate sample was counted three times (for a total of six counts per replicate per day). At test termination, a microscopic visual observation on the general health of the cells at each test level was made.
Cell density was determined using a haemocytometer. Biomass determinations were made using the cell volume endpoint using a Coulter Multisizer 3 at approximately 24, 48, 72, 96, and 120 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.0 μm to a higher threshold equivalent spherical diameter of approximately 12.6 μm. Three separate cell volume readings were made per sample. The readings for the blank replicates were used to correct for background in daily calculations. The corrected cell volumes were converted to volume x 10000/mL for statistical analysis and reporting.
Using the same data collection event, total cell counts were also determined at approximately 24, 48, 72, 96, and 120 hours. A correction factor of approximately 6X was then applied to these data to get an approximate cell density. The 6X correction factor was determined between the haemocytometer counts and Coulter Multisizer 3 counts on Day 0.
In addition, at test termination morphological observations were done on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus BHB-DO System Microscope (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck) using a Bright Line Haemocytometer Counting Chamber.

ENVIRONMENTAL PARAMETERS
At test initiation, a pH measurement was taken from a sample of the bulk preparation of each exposure concentration and the control. At the 72-hour observation period pH measurements were taken from composite samples of the control and exposure concentration solutions. Composite samples were prepared by withdrawing and combining 2.5 mL volumes from each inoculated replicate at each level. No 72-hour pH measurements were made on blank replicates. At test termination, a final pH measurement was taken from a pooled sample of the replicates with algae at each exposure concentration and the control and separately from each blank replicate. Incubator temperature was continuously monitored with a minimum/maximum thermometer probe placed in a representative vessel. Light intensity was measured at test initiation with a Dual Display Light Meter and was recorded at test initiation at each position where inoculated replicates were placed during the in-life phase.

TEST CONCENTRATIONS
The test concentrations selected for evaluating the effects of the test material on the growth of Anabaena flos-aquae were based on the results of a previous test using the same test species. This study was rejected by regulatory authorities based on the low growth, as well as variability in growth, during the first 96 hours of the test. This study was conducted as a repeat of the rejected study. The test levels were adjusted slightly from the original report in an attempt to generate a better dose-response. The target concentrations were set based on this preliminary information.

Reference substance (positive control):

no

Key result

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

23.3 - 47.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: The Logistic Sigmoid and Cumulative Normal models did not provide reliable estimates of the ErC50 values. Based on this, the ErC50 can be empirically determined to be between 23.3 and 47.2 mg/L.

Key result

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

23.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

120 h

Dose descriptor:

EC50

Effect conc.:

27.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 25.4 - 30.2 mg a.i./L (95% CL)

Key result

Duration:

120 h

Dose descriptor:

NOEC

Effect conc.:

23.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

CELL DENSITY
Cell density was not used as the primary biomass endpoint for this species based on the difficulties determining an accurate cell count for this species. However, using the same data collection event, total “cell counts” were also determined at approximately 24, 48, 72, 96, and 120 hours. The haemocytometer: Coulter Multisizer 3 correction factor of approximately 6X was then applied to these data to get an approximate cell density.

CELL VOLUME
Mean cell volumes at 72 hours were 1350, 1177, 1324, 1379, 1129, 31.5, and 1.82 x 10⁴ μm³/mL for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 2 % stimulation to 100 % inhibition of cell volume.
Mean cell volumes at 96 hours were 4044, 3550, 3928, 4381, 3460, 22.2, and 5.84 x 10⁴ μm³/mL for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 8 % stimulation to 100 % inhibition of cell volume.
Mean cell volumes at 120 hours were 7964, 7513, 8184, 8118, 7048, 19.0, and 0.290 x 10⁴ μm³/mL for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 3 % stimulation to 100 % inhibition of cell volume.

SPECIFIC GROWTH RATE (BASED ON CELL VOLUME)
Mean specific growth rates at 72 hours were 0.0514, 0.0495, 0.0510, 0.0517, 0.0489, -0.00744, and -0.0580 hour-1 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 1 % stimulation to 213 % inhibition of mean specific growth rate.
Mean specific growth rates at 96 hours were 0.0499, 0.0486, 0.0495, 0.0508, 0.0483, -0.00709, and -0.0396 hour-1 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 2 % stimulation to 179 % inhibition of mean specific growth rate.
Mean specific growth rates at 120 hours were 0.0456, 0.0452, 0.0458, 0.0458, 0.0446, -0.00962, and -0.0457 hour-1 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 0 % to 200 % inhibition of mean specific growth rate.

BIOMASS AREA (AREA UNDER THE GROWTH CURVE, BASED ON CELL VOLUME)
Mean biomass values at 72 hours were 28752, 25562, 28218, 27522, 22682, -1116, and -1824 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L/L test levels, respectively. Response relative to the controls ranged from 2 % to 106 % inhibition of biomass.
Mean biomass values at 96 hours were 92677, 81491, 90443, 95843, 76959, -1272, and -2532 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L/L test levels, respectively. Response relative to the controls ranged from 3 % stimulation to 103 % inhibition of biomass.
Mean biomass values at 120 hours were 235964, 213444, 234988, 245040, 202260, -1577, and -3257 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L/L test levels, respectively. Response relative to the controls ranged from 4 % stimulation to 101 % inhibition of biomass.

MORPHOLOGICAL OBSERVATIONS
Microscopic evaluation of cells at each test concentration and the control revealed no abnormal observations at test levels ≤ 23.3 mg/L. At both the 47.2 and 94.7 mg/L test levels there were few cells and the chains were short and not well developed.

Reported statistics and error estimates:

The data (untransformed, square root transformed for cell density, and the log transform for biomass and growth rate) were tested for normality using the Shapiro-Wilk’s Test (α = 0.01) and for homogeneity of variance using the Levene’s Test (α = 0.01). If the assumptions of normality and homogeneity were met for the untransformed data or one of the transformations, no-observed-effect concentrations (NOECs) were determined using a one-tailed Dunnett's mean comparison procedure (α = 0.05). If the assumptions of normality and/or homogeneity were not met and a non-parametric analysis was required, the ranks of the raw data values were determined and then the above analysis was performed on these ranks.
The 72-, 96-, and 120-hour ErC50 values (the concentration which reduced the algal growth rate by 50 % relative to the control), the 72-, 96-, and 120-hour EbC50 values (the concentration which reduced biomass by 50 % relative to the control), and the 72-, 96-, and 120-hour EC50 values (the concentration which reduced cell volume by 50 % relative to the control) were determined. Two sigmoid-shaped nonlinear models were used for calculation of EC50 values and 95 % confidence intervals, if possible. One model used to describe the response to increasing concentrations was the four-parameter logistic model (percent inhibition versus concentration). The second model used was the cumulative normal model (response compared to log concentration). The model that best fit the data was reported. These statistical analyses were conducted in SAS version 6.12 (SAS Institute, Cary, North Carolina).

Analytical Chemistry

The day 0 analysis of bulk dose solutions yielded values ranging from 91.8 to 101 % of the target concentration. The day 5 analysis of pooled, spent test solutions afforded values ranging from 87.8 to 96.5 % of the target concentration. A mean measured concentration was also calculated by averaging the day 0 bulk values with the day 5 pooled, spent values at each dose level. The mean measured concentration ranged from 89.9 to 99.0 % of target. None of the analyses of the AAM controls exhibited peaks eluting at the retention time of the analyte at concentrations exceeding the lowest level quantified (LLQ).

Water Quality Parameters

Temperatures during the exposure period ranged from 23.5 - 23.7 °C. Light intensity ranged from 2050 – 2400 lux. The pH values ranged from 7.3 to 3.3 at test initiation, from 7.6 to 3.5 in pooled replicates with algae on day 3, and from 9.3 to 3.5 in pooled replicates with algae and 7.3 to 3.5 in blank replicates without algae at test termination. The initial pH values and terminal pH values in replicates without algae (blanks) were negatively correlated with the test solution concentration (i.e., as test concentration increased pH values decreased). The values noted on days 3 and 5 in pooled samples of replicates with algae showed similar pH values at test levels <23.3 mg/L. The pH values at the two highest levels were in the range of 3.5 to 4.5.

Table 1: Mean Specific Growth Rate After 72, 96 and 120 Hours of Exposure

Mean measured conc. (mg/L)	Specific growth rate (hour^-1)
	Exposure hour
	72	72 hr % inh.	96	96 hr % inh.	120	120 hr % inh.
Medium control	0.0514	NA	0.0499	NA	0.0456	NA
3.10	0.0495	4	0.0486	3	0.0452	1
5.62	0.0510	1	0.0495	1	0.0458	0
11.6	0.0517	-1	0.0508	-2	0.0458	0
23.3	0.0489*	5	0.0483	3	0.0446	2
47.2	-0.00744*	114	-0.00709*	114	-0.00962*	121
94.7	-0.0580*	213	-0.0396*	179	-0.0457*	200

 * Significant difference from the controls; p ≤ 0.05, one-tailed Dunnett's t-test.

Table 2: Mean Biomass Area (area under the growth curve) at 72, 96 and 120 Hours of Exposure

Mean measured conc. (mg/L)	Biomass area (area under the growth curve)
	Exposure hour
	72	72 hr % inh.	96	96 hr % inh.	120	120 hr % inh.
Medium control	28752	NA	92677	NA	235964	NA
3.10	25562	11	81491	12	213444	10
5.62	28218	2	90443	2	234988	0
11.6	27522	4	95843	-3	245040	-4
23.3	22682*	21	76959*	17	202260*	14
47.2	-1116*	104	-1272*	101	-1577*	101
94.7	-1824*	106	-2532*	103	-3257*	101

* Significant difference from the controls; p ≤ 0.05, one-tailed Dunnett's t-test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 120-hour ErC50 was empirically determined to be between 23.3 and 47.2 mg a.i./L with a NOEC value of 23.3 mg a.i./L. In terms of biomass, the 120 hour EbC50 was determined to be 27.8 mg a.i./L, the corresponding NOEC was 23.3 mg a.i./L.
The 96-hour ErC50 was empirically determined to be between 23.3 and 47.2 mg a.i./L with a NOEC value of 23.3 mg a.i./L. In terms of biomass, the 96-hour EbC50 was determined to be 27.3 mg a.i./L, the corresponding NOEC was 11.6 mg a.i./L.

Executive summary:

The toxicity of the test material to the freshwater alga, Anabaena flos-aquae, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), OECD 201 and EU Method C.3.

The algae were exposed to nominal concentrations of test material of 0 (medium control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L, under static conditions, for a period of 120 hours. Three replicates at each test level and six replicates at the medium control level were inoculated with approximately 20 000 cells/mL. The additional replicate at each test and control level was not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass.

At approximately 24, 48, 72, 96, and 120 hours, samples were withdrawn from each replicate for determination of particle counts and cell volume. Each replicate sample was counted three times (for a total of six counts per replicate per day). At test termination, a microscopic visual observation on the general health of the cells at each test level will be made.

Mean cell volumes at 120 hours were 7964, 7513, 8184, 8118, 7048, 19.0, and 0.290 x 10^4 μm³/mL for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 3 % stimulation to 100 % inhibition of cell volume. Mean biomass values at 120 hours were 235964, 213444, 234988, 245040, 202260, -1577, and -3257 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L/L test levels, respectively. Response relative to the controls ranged from 4 % stimulation to 101 % inhibition of biomass.

Under the conditions of the study, the 120-hour ErC50 was empirically determined to be between 23.3 and 47.2 mg a.i./L with a NOEC value of 23.3 mg a.i./L. In terms of biomass, the 120 hour EbC50 was determined to be 27.8 mg a.i./L, the corresponding NOEC was 23.3 mg a.i./L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 February 2002 to 5 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 123-2 (Growth and Reproduction of Aquatic Plants (Tier 2))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

At test initiation and test termination (120 hours), samples were removed from each test solution and the controls and analysed for test material. Samples analysed on day 0 were removed from the test solutions in the 1000 and 2000 mL flasks prior to filling the individual test flasks. Samples analysed at 120 hours of exposure were removed from the composite of the three replicate vessels for each treatment and control.
At 120 hours of exposure, a sample was removed from the replicate flask (D) of the 25 mg a.i./L test concentration which did not contain algae. The result of this analysis was compared with that obtained for the 120-hour analysis of the 25 mg a.i./L solution containing algae to assess the impact that algae had on the test material concentration.
Three quality control (QC) samples containing the test material was prepared at each sampling interval using freshly-prepared algal growth medium at nominal concentrations similar to the exposure concentration range and remained with the corresponding set of exposure solution samples throughout the chemical analysis. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.

Vehicle:

yes

Details on test solutions:

A 100 mg a.i./L primary stock solution was prepared by placing 0.2114 g of test material (0.1998 g as active ingredient) in a 2000-mL volumetric flask and diluting to volume with algal assay procedure (AAP) medium containing 0.1 mL/L of dimethylformamide (DMF). The resulting stock solution was observed to be clear and colourless with no undissolved test material. Test solutions were prepared from dilutions of the 100 mg a.i./L primary stock solution.
A solvent control was also prepared by diluting 0.10 mL of DMF to volume with AAP medium in a 1000-mL flask. The concentration of DMF in the solvent control was equal to the concentration of DMF present in the test solutions (0.10 mL/L). Additional untreated AAP medium was used for the control.

Test organisms (species):

Navicula pelliculosa

Details on test organisms:

TEST ORGANISM
- Strain: 1530.45 (Class Bacillariophyceae)
Stock cultures were grown in 125 mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The cultures were maintained within the following conditions since the last transfer: shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C, and continuous illumination at the surface of the medium with an intensity of approximately 300 to 500 footcandles (3200 to 5400 lux). Temperature was controlled using an environmental chamber. Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker.
The inoculum used to initiate the toxicity test with test material was taken from a stock culture that had been transferred to fresh medium 6 days prior to test initiation.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

23 - 24 °C

pH:

3.6 - 7.1 (at test initiation); 3.6 - 9.7 (test termination)

Nominal and measured concentrations:

6.3, 13, 25, 50 and 100 mg a.i./L (nominal)
6.0, 12, 23, 48 and 100 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL flasks filled with 100 mL test solution
- Aeration: shaking rate was maintained throughout the exposure at a constant rate of 100 rpm.
- Initial cell density: 1.0 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
Replicate 250-mL flasks, three per treatment level and the controls, were conditioned prior to use by rinsing with the appropriate test solution. One hundred millilitres of the appropriate test solution was then placed in each replicate flask. Three flasks for each set of controls were maintained under the same conditions as the treatment level vessels but contained no test material. All test vessels were fitted with stainless steel caps which permitted gas exchange.
In order to estimate the impact that the presence of algal biomass had on the test material concentration, an additional replicate flask (D) of the 25 mg a.i./L (nominal) treatment level was prepared. This flask, which was not inoculated with algae, was analysed at 120 hours of exposure for test material concentration. The result of this analysis was compared with the results for the 25 mg a.i./L solution containing algae.

GROWTH MEDIUM
- Standard medium used: Yes. The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionised water. The composition of the AAP medium was as follows: NaNO₃ (25.5 mg/L); MgCl₂.6H₂O (12.16 mg/L); CaCl₂.2H₂O (4.41 mg/L); MgSO₄.7H₂O (14.7 mg/L); K₂HPO₄.3H₂O (1.368 mg/L); NaHCO₃ (15.0 mg/L); H₃BO₃ (185.5 μg/L); Na₂SeO₄ (1.88 μg/L); MnCl₂.4H₂O (415.4 μg/L); ZnCl₂ (3.270 μg/L); CoCl₂.6H₂O (1.43 μg/L); CuCl₂.2H₂O (0.012 μg/L); Na₂MoO₄.2H₂O (7.26 μg/L); FeCl₃.6H₂O (159.8 μg/L); Na₂EDTA.2H₂O (300.0 μg/L). If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl
- Photoperiod: continuous illumination
- Light intensity and quality: Light intensity of the test area ranged from 375 to 500 footcandles. The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 57 to 88 μE/m²/s.

EFFECT PARAMETERS MEASURED
At each 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment levels and control using a haemocytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval. At test termination, due to the habit of Navicula pelliculosa cells to clump, the solutions were vigorously pipetted multiple times to break up clumped cells and achieve a homogeneous suspension prior to removing a sample for cell counts.

ENVIRONMENTAL PARAMETERS
Temperature was measured continuously with a Fisher minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured with a General Electric Type 214 light meter at 0 hour and at each 24-hour interval during the exposure period. The PAR of the test area was measured at test initiation using a Licor photometer (model LI-190SA). Test flasks were randomly placed on the shaking table at test initiation based upon computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers.
Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period. Measurements at test initiation were conducted on the test solutions remaining in the mixing vessels after the individual test flasks had been filled. At test termination, after cell counts were completed, the three replicate vessels for the test concentration and the controls were composited and a portion of the composite solution was transferred to a 100-mL beaker for pH and conductivity measurements. Test solution pH was measured with a Jenco Model 60 pH meter and conductivity was measured with a Yellow Springs Instrument (YSI) Model #33 salinity-conductivity-temperature meter.

PRELIMINARY TESTING
A preliminary range-finding exposure was conducted at the test laboratory at nominal test material concentrations of 0.10, 1.0, 10 and 100 mg a.i./L, a control and a solvent control. Two exposure vessels were established for each concentration and the controls. Following 120 hours of exposure, cell densities in the 0.10, 1.0, 10 and 100 mg a.i./L treatment levels averaged 136, 171, 194 and 0 x 10⁴ cells/mL, respectively. The control and solvent control averaged 140 and 199 x 10⁴ cells/mL. Based on these data, nominal test material concentrations of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 3.7 - 140 mg a.i./L (95% CL)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Prior to the test termination observation interval, the test solutions were vigorously pipetted multiple times to disperse clumped cells and provide a homogeneous suspension for counting. At test termination (120 hours), cells exposed to the treatment levels tested and the controls were observed to be normal.

CELL DENSITY
The 120-hour cell density in the control and solvent control averaged 237 and 205 x 10⁴ cells/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control cell density; therefore control and solvent control data were pooled (pooled control = 221 x 10⁴ cells/mL). Cell density in the 6.0, 12, 23, 48 and 100 mg a.i./L treatment levels averaged 188, 160, 174, 0 and 0 x 10⁴ cells/mL, respectively. Based on the results of Chi-square and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. A significant reduction in cell density was observed in the 12, 23, 48 and 100 mg a.i./L treatment levels as compared to the pooled control data.

BIOMASS
The total biomass in the control and solvent control averaged 21.4 and 17.1 x 10⁴ cells.days/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control biomass; therefore control and solvent control data were pooled (pooled control = 19.2 x 10⁴ cells.days/mL). Total biomass in the 6.0, 12, 23, 48 and 100 mg a.i./L treatment levels averaged 16.3, 11.9, 13.1, -1.1 and -1.8 cells.days/mL, respectively. Statistical analysis (Williams' Test) determined a significant difference in biomass in the 12, 23, 48 and 100 mg a.i./L treatment levels when compared to the biomass in the pooled control (19.2 x 10⁴ cells.days/mL).

GROWTH RATES
The 0- to 72-hour growth rate in the control and solvent control averaged 1.12 and 1.01 days^-1, respectively. Statistical analysis (t-Test) determined a significant difference between control and solvent control growth rate; therefore the treatment data were compared with the solvent control data. The 0- to 72-hour growth rate in the 6.0, 12, 23, 48 and 100 mg a.i./L treatment levels averaged 1.03, 0.87, 1.03, -0.35 and -0.47 days^-1, respectively. Statistical analysis (Williams’ Test) determined a significant reduction in treatment levels 48 and 100 mg a.i./L when compared to the growth rate of the solvent control.

Reported statistics and error estimates:

A t-Test was conducted to statistically compare the 120-hr cell density of the control to the solvent control. If no significant difference was determined, control and solvent control data were pooled for comparison to treatment data. If a difference was determined between the control and solvent control data, the solvent control was used for comparison with the treatment data. % inhibition of the treatment data was calculated relative to the appropriate control data.
Based on the results of statistical analysis performed for 120-hr cell density, 72-hr total biomass and average growth rate the NOEC was determined. The data were first checked for normality using Shapiro-Wilks' Test and Chi-square Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the tests for homogeneity and normality, then Williams’ Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis’ Test was used to determine the NOEC. All statistical determinations were made at the 95 % level of certainty, except in the case of Shapiro-Wilks', Chi-square and Bartlett's Tests, where the 99 % level of certainty was applied.
The EC25 and EC50 values and the 95 % confidence limits were calculated. The EC value was determined by linear regression of response versus exposure concentration over the range of test concentrations where a clear exposure-response relationship was observed. Four linear regressions were estimated based on (a) untransformed data, (b) untransformed response vs. logarithm-transformed concentration, (c) probit-transformed response vs. untransformed concentration, and (d) probit-transformed response vs. logarithm-transformed concentration. The regression that best fit the data was selected based on the highest coefficient of determination (r²). This regression equation was then applied to estimate the EC values and their 95 % confidence limits, using the method of inverse prediction.

Analytical Chemistry

Based on analyses at test initiation and termination, measured concentrations ranged from 92 to 100 % of nominal and defined the mean measured treatment levels as 6.0, 12, 23, 48 and 100 mg a.i./L. The analytical result of the 120-hour sample from the 25 mg a.i./L nominal treatment level, with algae present, was 23 mg a.i./L. The equivalent test solution without algae present resulted in a recovery of 23 mg a.i./L and demonstrated that the presence of algae had no impact on the concentration of test material in the test solution. Analysis of the quality control samples resulted in recoveries which were consistent with the pre-determined recovery range and ranged from 100 to 111 % (N = 6) of the nominal fortified levels (6.00 to 100 mg a.i./L). Based on the results of these analyses, it was established that the appropriate precision and quality control was maintained during the analysis of the exposure solutions.

Water Quality Parameters

The pH of the test solutions ranged from 3.6 to 7.1 at test initiation and was inversely proportional to the test concentration. At test termination, the test solution pH ranged from 3.6 to 9.7. Conductivity measured at test initiation and termination in the treatment and control solutions ranged from 90 to 180 μmhos/cm. Continuous temperature monitoring established that the temperature ranged from 23 to 24 °C during the test period. Light intensity of the test area ranged from 375 to 500 footcandles. The PAR of the test area measured at test initiation ranged from 57 to 88 μE/m²/s. The shaking rate was maintained throughout the exposure at a constant rate of 100 rpm.

Table 1: Mean Cell Density After 24, 48, 72, 96 and 120 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Cell density (x 10⁴ cells/mL)
	Observation interval (hours)
	24	48	72	96	120	Percent inhibition*
Control	2.00	8.50	27.30	63.00	237.00	NA
Solvent control	1.83	8.17	19.90	63.00	205.00	NA
6.0	2.42	5.92	21.30	49.00	188.00	15
12	2.33	5.83	13.00	37.00	160.00**	28
23	1.33	3.67	21.10	46.00	174.00**	21
48	0.67	0.58	0.30	0.00	0.00**	100
100	0.25	0.33	0.10	0.00	0.00**	100

*Percent inhibition relative to the pooled control (0 - 120 hour cell density 221 x 10⁴ cells/mL)

**Significantly reduced as compared to the pooled control, based on Williams’ Test.

Table 2: Mean Calculated Biomass (area under the growth curve) After 24, 48 and 72 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Biomass (x 10⁴ cells.days/mL)
	Observation interval (hours)
	0 - 24	24 - 48	48 - 72	Total biomass	Percent inhibition*
Control	0.55	3.47	17.3	21.4	NA
Solvent control	0.46	3.26	13.4	17.1	NA
6.0	0.78	2.58	13.0	16.3	15
12	0.74	2.52	8.7	11.9**	38
23	0.18	1.22	11.7	13.1**	32
48	-0.18	-0.31	-0.6	-1.1**	106
100	-0.41	-0.58	-0.8	-1.8**	109

*Percent inhibition relative to the pooled control (0 - 72 hour biomass 19.2 x 10⁴ cells.days/mL)

**Significantly reduced as compared to the pooled control, based on Williams’ Test.

Table 3: Mean Calculated Growth Rates After 24, 48 and 72 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Growth rate (days^-1)
	Observation interval (hours)
	0 - 24	0 - 48	0 - 72	Percent inhibition*
Control	0.55	1.08	1.12	NA
Solvent control	0.47	1.06	1.01	NA
6.0	0.55	0.92	1.03	4
12	0.73	0.91	0.87	19
23	0.24	0.67	1.03	4
48	-0.42	-0.36	-0.35**	133
100	-0.26	-0.60	-0.47**	144

*Percent inhibition relative to the solvent control

**Significantly reduced as compared to the solvent control, based on Williams’ Test.

Validity criteria fulfilled:

yes

Conclusions:

Based on cell density, the 120-hour NOEC was determined to be 6.0 mg a.i./L. The 120-hour EC25 and EC50 value (corresponding 95 % confidence intervals) were calculated to be 11 mg a.i./L (2.6 to 38 mg a.i./L) and 22 mg a.i./L (6.0 to 81 mg a.i./L), respectively. Based on cell biomass, the NOEC was determined to be 6.0 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 18 mg a.i./L (5.4 to 59 mg a.i./L). Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 23 mg a.i./L. The 72-hour ErC50 (corresponding 95 % confidence intervals) was calculated to be 21 mg a.i./L (3.7 to 140 mg a.i./L).

Executive summary:

The toxicity of the test material to the freshwater alga, Navicula pelliculosa, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3.

Based on the results of preliminary testing, nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent control was also prepared by diluting DMF in AAP medium. Additional untreated AAP medium was used for the control. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Within 45 minutes of preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 0.263 mL of an inoculum of Navicula pelliculosa cells, at a density of 380 x 10⁴cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell density of approximately 1.0 x 10⁴cells/mL.

At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval. At test termination, due to the habit of Navicula pelliculosa cells to clump, the solutions were vigorously pipetted multiple times to break up clumped cells and achieve a homogeneous suspension prior to removing a sample for cell counts.

Temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period.

Prior to the test termination observation interval, the test solutions were vigorously pipetted multiple times to disperse clumped cells and provide a homogeneous suspension for counting. At test termination (120 hours), cells exposed to the treatment levels tested and the controls were observed to be normal.

Under the conditions of the study a significant reduction in both cell density, and biomass, was observed in the 12, 23, 48 and 100 mg a.i./L treatment levels as compared to the pooled control data. In addition, statistical analysis determined a significant reduction in treatment levels 48 and 100 mg a.i./L when compared to the growth rate of the solvent control.

Based on cell density, the 120-hour NOEC was determined to be 6.0 mg a.i./L. The 120-hour EC25 and EC50 value (corresponding 95 % confidence intervals) were calculated to be 11 mg a.i./L (2.6 to 38 mg a.i./L) and 22 mg a.i./L (6.0 to 81 mg a.i./L), respectively. Based on cell biomass, the NOEC was determined to be 6.0 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 18 mg a.i./L (5.4 to 59 mg a.i./L). Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 23 mg a.i./L. The 72-hour ErC50 (corresponding 95 % confidence intervals) was calculated to be 21 mg a.i./L (3.7 to 140 mg a.i./L), NOEC = 23 mg a.i./L.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 March 2002 to 19 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 123-2 (Growth and Reproduction of Aquatic Plants (Tier 2))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

At test initiation and test termination (120 hours), samples were removed from each test solution and the controls and analysed for test material. Samples analysed on day 0 were removed from the test solutions in the 1000 or 2000 mL mixing vessels prior to filling the individual test flasks. Samples analysed at 120 hours of exposure were removed from the composite of replicate solutions from each treatment and control.
At 120 hours of exposure, a sample was removed from the replicate flask (D) of the 25 mg a.i./L test concentration which did not contain algae. The result of this analysis was compared with that obtained for the 120-hour analysis of the 25 mg a.i./L solution containing algae to assess the impact that algae had on the test material concentration.
Three quality control (QC) samples containing the test material was prepared at each sampling interval using freshly-prepared algal growth medium at nominal concentrations similar to the exposure concentration range and remained with the corresponding set of exposure solution samples throughout the chemical analysis. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.

Vehicle:

yes

Details on test solutions:

A 100 mg a.i./L primary stock solution was prepared by placing 0.2115 g of test material (0.1999 g as active ingredient) in a 2000 mL volumetric flask and diluting to volume with artificially enriched seawater (AES) medium containing 0.1 mL/L of dimethylformamide (DMF). The resulting stock solution was observed to be clear and colourless with no visible undissolved test material. Test solutions were prepared from dilutions of the 100 mg a.i./L primary stock solution.
A solvent control was also prepared by diluting 0.10 mL of DMF to volume with AES medium in a 1000 mL flask. The concentration of DMF in the solvent control was equal to the concentration of DMF present in the test solutions (0.10 mL/L). Additional untreated AES medium was used for the control.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Strain: strain CCMP 1332 (Class Bacillariophyceae)
Stock cultures were grown in 125 mL glass flasks each containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The cultures were maintained within the following conditions after the last transfer: shaking rate of 60 ± 10 rpm, a temperature of 20 ± 1 °C, and continuous illumination at the surface of the medium with an intensity of approximately 300 to 500 footcandles (3200 to 5400 lux). Temperature was controlled using an environmental chamber. Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker.
The inoculum used to initiate the toxicity test with test material was taken from a stock culture that had been transferred to fresh medium six days prior to test initiation.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

120 h

Test temperature:

20 - 21 °C

pH:

7.0 - 8.0 (at test initiation); 8.5 - 8.7 (at test termination)

Salinity:

Salinity of the medium was within the range of 30 ± 2 g/L.

Nominal and measured concentrations:

6.3, 13, 25, 50 and 100 mg a.i./L (nominal)
6.2, 13, 25, 50 and 100 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL vessels containing 100 mL of test solution. All test vessels were fitted with stainless steel caps which permitted gas exchange.
- Aeration: the vessels were agitated on an orbital shaker table set at 60 ± 10 rpm
- Initial cell density: Approximately 1.0 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3
Replicate 250 mL flasks, three per treatment level and the controls, were conditioned prior to use by rinsing with the appropriate test solution. One hundred millilitres of the appropriate test solution was then placed in each replicate flask. Two sets of control flasks (control and solvent control) were maintained under the same conditions as the treatment level vessels but contained no test material.
In order to estimate the impact that the presence of algal biomass had on the test material concentration, an additional replicate flask (D) of the 25 mg a.i./L (nominal) treatment level was prepared. This flask, which was not inoculated with algae, was analysed at 120 hours of exposure for test material concentration. The result of this analysis was compared with the results for the 25 mg a.i./L solution containing algae.

GROWTH MEDIUM
- Standard medium used: yes. The culture medium used was Artificially Enriched Seawater (AES) medium prepared with sterile, filtered, natural seawater. The composition of the AES medium was as follows: FeCl₃.6H₂O (7.2 mg/L); MnCl₂.4H₂O (2.16 mg/L); ZnSO₄.7H₂O (0.777 mg/L); CuSO₄.5H₂O (0.00236 mg/L); CoCl₂.6H₂O (0.00606 mg/L); Na₂MoO₄.2H₂O (0.0528 µg/L); Na₂SeO₄ (1.88 µg/L); H₃BO₃ (2.10 mg/L); K₃PO₄ (3.0 mg/L); NaNO₃ (50 mg/L); NaSiO₃.9H₂O (20 mg/L); Thiamine hydrochloride.1/2H₂O (0.2575 mg/L); Biotin (0.050 µg/L); Vitamin B₁₂ (0.50 µg/L); Na₂EDTA.2H₂O (0.3848 mg/L).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the culture medium was adjusted to pH 8.0 ± 0.1 with either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide, if necessary.
- Photoperiod: continuous light
- Light intensity and quality: Light intensity of the test area ranged from 300 to 400 footcandles. The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 50 to 57 μE/m²/s.
- Salinity: Salinity of the medium was within the range of 30 ± 2 g/L.

EFFECT PARAMETERS MEASURED
At each 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval.

ENVIRONMENTAL PARAMETERS
Temperature was measured continuously with a Fisher minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured with a General Electric Type 214 light meter at 0 hour and at each 24-hour interval during the exposure period. The PAR of the test area was measured at test initiation using a Licor photometer (model LI-190SA). Test flasks were randomly placed on the shaking table at test initiation based upon computer generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers.
Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period. Measurements at test initiation were conducted on the test solutions remaining in the mixing vessels after the individual test flasks had been filled. At test termination, after cell counts were completed, the three replicate vessels for the test concentration and the control were composited and a portion of the composite solution was transferred to a 100-mL beaker for pH and conductivity measurements. Test solution pH was measured with a Jenco Model 60 pH meter and conductivity was measured with a Yellow Springs Instrument (YSI) Model #33 salinity-conductivity-temperature meter.

PRELIMINARY TESTING
Preliminary range-finding exposure was conducted at the test laboratory at nominal test material concentrations of 0.10, 1.0, 10 and 100 mg a.i./L, a control and a solvent control. Two exposure vessels were established for each concentration and the controls. Following 120 hours of exposure, cell densities in the 0.10, 1.0, 10 and 100 mg a.i./L treatment levels averaged 136, 128, 98 and 115 x 10⁴ cells/mL, respectively. The control and solvent control averaged 103 and 135 x 10⁴ cells/mL. Based on these data, nominal test material concentrations of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Cells exposed to the 6.2, 13, 25, 50 and 100 mg a.i./L treatment levels and the controls were observed to be normal.

CELL DENSITY
The 120-hour cell density in the control and solvent control culture averaged 94 and 112 x 10⁴ cells/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control cell density; therefore, control and solvent control data were pooled (pooled control = 103 x 10⁴ cells/mL). Cell density in the 6.2, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 62, 112, 94, 103 and 97x 10⁴ cells/mL, respectively. All treatments were determined to be similar to the pooled control data.

CELL BIOMASS
The total biomass in the control and solvent control averaged 18.5 and 23.3 x 10⁴ cells.days/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control biomass; therefore, control and solvent control data were pooled (pooled control = 20.9 x 10⁴ cells.days/mL). Total biomass in the 6.2, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 24.1, 20.6, 14.3, 14.1 and 8.7 cells.days/mL, respectively. Statistical analysis (Williams' Test) determined a significant difference in biomass in the 25, 50 and 100 mg a.i./L treatment levels when compared to the biomass in the pooled control (20.9 x 10⁴ cells.days/mL).

GROWTH RATE
The 0- to 72-hour growth rate in the control and solvent control averaged 1.01 and 1.10 days^-1, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control growth rate; therefore, the treatment data were compared with the pooled control data (1.06 days^-1). The 0- to 72-hour growth rate in the 6.2, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 1.06, 1.04, 0.91, 0.90 and 0.78 days^-1, respectively. Statistical analysis (Williams’ Test) determined a significant reduction in treatment levels 25, 50 and 100 mg a.i./L when compared to the growth rate of the pooled control.

Reported statistics and error estimates:

A t-test was conducted to statistically compare the 24-, 48-, 72-, 96- and 120-hour cell density as well as the 72-hour total biomass and growth rate data of the control to the solvent control. If no significant difference was determined, control and solvent control data were pooled for comparison to treatment data. If a difference was determined, the solvent control was used for comparison with the treatment data. % inhibition of the treatment data was calculated relative to the appropriate control data.
Based on the results of statistical analysis performed for 120-hour cell density, 72-hour total biomass and average growth rate, the NOEC was determined. The data were first checked for normality using Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the tests for homogeneity and normality, then Williams’ Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis’ Test was used to determine the NOEC. All statistical determinations were made at the 95 % level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99 % level of certainty was applied.
The EC25 and EC50 values and the 95 % confidence limits were calculated for cell densities after 24, 48, 72, 96 and 120 hours of exposure. Additionally, EC50 values were calculated for 72-hour EbC50 and ErC50. The EC value was determined by linear regression of response versus exposure concentration over the range of test concentrations where a clear exposure-response relationship was observed. Four linear regressions were estimated based on (a) untransformed data, (b) untransformed response vs. logarithm-transformed concentration, (c) probit-transformed response vs. untransformed concentration, and (d) probit-transformed response vs. logarithm-transformed concentration. The regression that best fit the data was selected based on the highest coefficient of determination (r²).

Analytical Chemistry

Based on analysis at test initiation and termination, measured concentrations ranged from 96 to 100 % of nominal concentration and defined the mean measured treatment levels as 6.2, 13, 25, 50 and 100 mg a.i./L. The analytical result of the 120-hour sample from the 25 mg a.i./L nominal treatment level, with algae present, was 25 mg a.i./L. The equivalent test solution without algae present resulted in a recovery of 24 mg a.i./L and demonstrated that the presence of algae had no impact on the concentration of test material in the test solution. Analysis of the quality control samples resulted in recoveries which were consistent with the pre-determined recovery range and ranged from 98.5 to 105 % (N = 6) of the nominal fortified levels (6.00 to 100 mg a.i./L). Based on the results of these analyses, it was established that the appropriate precision and quality control was maintained during the analysis of the exposure solutions.

Water Quality Parameters

The pH of the test solutions ranged from 7.0 to 8.0 at test initiation. At test termination, the pH ranged from 8.5 to 8.7. Conductivity measured at test initiation and termination in the treatment and control solutions ranged from 38 000 to 39 000 μmhos/cm. Continuous temperature monitoring established that the temperature ranged from 20 to 21 °C during the test period. Light intensity of the test area ranged from 300 to 400 footcandles. The PAR of the test area measured at test initiation ranged from 50 to 57 μE/m²/s. The shaking rate was maintained throughout the exposure at a constant rate of 60 rpm.

Table 1: Mean Cell Density After 24, 48, 72, 96 and 120 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Cell density (x 10⁴ cells/mL)
	Observation interval (hours)
	24	48	72	96	120	Percent inhibition*
Control	3.42	6.25	22.6	45	94	NA
Solvent control	2.42	8.75	29.3	44	112	NA
6.2	3.50	11.17	24.1	62	62	40
13	2.17	8.92	24.1	52	112	-9
25	2.08	7.00	15.6	36	94	9
50	2.67	5.92	16.1	43	103	0
100	1.83	4.08	10.7	30	97	6

 *Percent inhibition relative to the pooled control (0 - 120 hour cell density 103 x 10⁴ cells/mL)

Table 2: Mean Calculated Biomass (area under the growth curve) After 24, 48 and 72 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Biomass (x 10⁴ cells.days/mL)
	Observation interval (hours)
	0 - 24	24 - 48	48 - 72	Total biomass	Percent inhibition*
Control	1.30	3.50	13.7	18.5	NA
Solvent control	0.76	4.19	18.4	23.3	NA
6.2	1.34	5.78	17.0	24.1	-15
13	0.63	4.15	15.8	20.6	1
25	0.58	3.23	10.5	14.3**	32
50	0.89	3.01	10.2	14.1**	33
100	0.45	1.79	6.5	8.7**	58

*Percent inhibition relative to the pooled control (0 - 72 hour biomass 20.9 x 10⁴ cells/mL)

**Significantly reduced as compared to the pooled control, based on Williams’ Test.

Table 3: Mean Calculated Growth Rates After 24, 48 and 72 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Growth rate (days^-1)
	Observation interval (hours)
	0 - 24	0 - 48	0 - 72	Percent inhibition*
Control	1.13	0.92	1.01	NA
Solvent control	0.82	1.06	1.10	NA
6.2	1.09	1.20	1.06	0
13	0.71	1.09	1.04	2
25	0.66	0.98	0.91**	14
50	0.81	0.89	0.90**	15
100	0.47	0.68	0.78**	26

*Percent inhibition relative to the pooled control (0 - 72 hour growth rate 1.06 days^-1)

**Significantly reduced as compared to the pooled control, based on Williams’ Test.

Validity criteria fulfilled:

yes

Conclusions:

In terms of cell densities, all treatments were determined to be similar to the pooled control data, therefore the 120-hour NOEC was determined to be 100 mg a.i./L. The 120-hour EC25 and EC50 values were empirically estimated to be > 100 mg a.i./L, the highest mean measured concentration tested. The 72-hour NOEC for total biomass was determined to be 13 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 77 mg a.i./L (13 to 1000 mg a.i./L). Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 13 mg a.i./L. The 72-hour ErC50 was empirically estimated to be >100 mg a.i./L, the highest mean measured concentration tested.

Executive summary:

The toxicity of the test material to the marine diatom, Skeletonema costatum, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3.

During the study, the organisms were exposed to nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L, under static conditions, for 120 hours. A solvent (DMF) control and untreated control (artificially enriched seawater) were included. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Approximately one hour after preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 0.938 mL of an inoculum of Skeletonema costatum cells, at a density of 107 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell density of approximately 1.0 x 10⁴ cells/mL.

At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval. Furthermore, temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period.

Under the conditions of the test, the findings for cell density showed all treatments to be similar to the pooled control data, therefore the 120-hour NOEC was determined to be 100 mg a.i./L. The 120-hour EC25 and EC50 values were empirically estimated to be > 100 mg a.i./L, the highest mean measured concentration tested. Statistical analysis of the cell biomass data determined a significant difference in biomass in the 25, 50 and 100 mg a.i./L treatment levels when compared to the biomass in the pooled control (20.9 x 10⁴ cells·days/mL). Based on these results, the 72-hour NOEC for total biomass was determined to be 13 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 77 mg a.i./L (13 to 1000 mg a.i./L). Statistical analysis of the growth data determined a significant reduction in treatment levels 25, 50 and 100 mg a.i./L when compared to the growth rate of the pooled control. Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 13 mg a.i./L. The 72-hour ErC50 was empirically estimated to be >100 mg a.i./L, the highest mean measured concentration tested.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

29 November 2001 to 9 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA OPP 123-2 (Aquatic plant growth)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

yes

Details on sampling:

At test initiation (0 hour) and test termination (96 hours), a single sample was removed from each test concentration and the controls and analysed for test material residue. Samples analysed at 0 hour were removed from the test and control solutions in the 500, 1000 and 2000 mL volumetric flasks prior to division into the replicate test vessels. Samples analysed at 96 hours were removed from individually composited replicate solutions of the treatment levels and controls. At 96 hours of exposure, a sample was also removed from the replicate flask (D) of the 25 mg a.i./L test concentration which did not contain algae. The result of this analysis was compared with that obtained for the 96-hour analysis of the 25 mg a.i./L solution containing algae to assess the impact that algae had on the test material concentration.
In addition, three quality control (QC) samples were prepared at each sampling interval at nominal test material concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.

Vehicle:

yes

Details on test solutions:

A 100 mg a.i./L primary stock solution was prepared by placing 0.2118 g of test material (0.2002 g a.i.) in a 2000-mL volumetric flask and diluting to volume with sterile AAP medium containing 0.10 mL/L of dimethyl formamide (DMF). The resulting stock solution was observed to be clear and colourless with a small amount of fine particulate matter suspended in solution.
A solvent control solution was prepared with AAP medium containing 0.10 mL/L DMF. Additional untreated AAP medium was used to prepare the control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 1648 (Class Chlorophyceae)
- Method of cultivation: Algae were maintained in stock culture at the test laboratory. Stock cultures were grown in 125-mL glass flasks each containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C and continuous illumination at the surface of the medium with an intensity of approximately 300 to 500 footcandles (3200 to 5400 lux). Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test with test material was taken from a stock culture that had been transferred to fresh medium three days before testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23 - 24 °C

pH:

3.5 - 7.5 (at test initiation); 3.5 - 9.8 (at 96 hours of exposure)

Nominal and measured concentrations:

0, 6.3, 13, 25, 50, 100 mg a.i./L (nominal)
0, 5.6, 12, 23, 46, 94 mg a.i./L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 250-mL Erlenmeyer flasks. All flasks were conditioned prior to use by rinsing with the appropriate exposure or control solution. One hundred millilitres of the appropriate exposure or control solution was then placed in each replicate flask. All test vessels were fitted with stainless steel caps which permit gas exchange.
- Aeration: An orbital shaker table provided a shaking rate of 100 ± 10 rpm.
- Initial cell density: Approximately 1.0 x 10⁴ cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
Three replicate flasks per treatment level (A, B and C) and the controls were prepared. In order to estimate the impact that the presence of algal biomass had on the test material concentration, one additional replicate flask (D) of the 25 mg a.i./L (nominal) treatment level was prepared and maintained during the 96-hour test. This flask, which was not inoculated with algae, was analysed at 96 hours for test material concentration.

GROWTH MEDIUM
- Standard medium used: Yes. The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionised water. The composition of the AAP medium was as follows: NaNO₃ (25.5 mg/L); MgCl₂.6H₂O (12.16 mg/L); CaCl₂.2H₂O (4.41 mg/L); MgSO₄.7H₂O (14.7 mg/L); K₂HPO₄.3H₂O (1.368 mg/L); NaHCO₃ (15.0 mg/L); H₃BO₃ (185.5 μg/L); Na₂SeO₄ (1.88 μg/L); MnCl₂.4H₂O (415.4 μg/L); ZnCl₂ (3.270 μg/L); CoCl₂.6H₂O (1.43 μg/L); CuCl₂.2H₂O (0.012 μg/L); Na₂MoO₄.2H₂O (7.26 μg/L); FeCl₃.6H₂O (159.8 μg/L); Na₂EDTA.2H₂O (300.0 μg/L). If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: If required, pH was adjusted to 7.5 ± 0.1 with 0.1 N NaOH or 0.1 N HCl
- Photoperiod: continuous illumination
- Light intensity and quality: Light intensity of the test area ranged from 300 to 420 footcandles (3200 to 4500 lux) throughout the exposure period. The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 35 to 61 μE/m²/s.

EFFECT PARAMETERS MEASURED
At each 24-hour interval, cell counts were conducted on the three replicate vessels (A, B and C) of the treatment and control vessels using a haemocytometer (Neubauer Improved) and compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were also made and recorded at each 24-hour interval.

ENVIRONMENTAL PARAMETERS
Temperature was measured continuously with a Fisher minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured with a General Electric Type 214 light meter at 0 hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a Licor photometer (model #LI-190SA). Test flasks were randomly placed on the shaking table at test initiation based on computer-generated random numbers. Following each observation interval, the test flasks were placed in new random positions.
Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 96-hour exposure period. Measurements at test initiation were conducted on the test solution in the 500, 1000 and 2000-mL flasks prior to division into the individual test flasks. At test termination, after cell counts were completed, the replicate solutions (A, B and C) for each treatment and the controls were respectively composited and a portion of the composite solution was transferred to a 100-mL beaker for pH and conductivity measurements. Test solution pH was measured with a Jenco Model 60 pH meter, and conductivity was measured with a Yellow Springs Instrument (YSI) Model #33 salinity-conductivity-temperature meter.

RECOVERY FOR ALGISTATIC/ALGICIDAL PROPERTIES
A composite sample of three replicate vessels (A, B and C) was removed from the 100 mg a.i./L test concentration. The sample was then diluted with freshly prepared AAP medium to prepare a subculture with a nominal concentration of 6.3 mg a.i./L. The performance of this subculture was used to determine if the effects of the test material on the alga were algistatic, in which case cells would resume growth in the subculture, or algicidal, in which case no growth would occur in the subculture. The subculture was incubated for six days under conditions consistent with those maintained during the definitive exposure. During this period, the subculture was microscopically examined every other day to determine whether or not cell growth had resumed. The subculture was discontinued after a substantial increase in cell density (i.e. >10X) was observed.

PRELIMINARY TEST
A preliminary exposure was conducted at the test laboratory at nominal test material concentrations of 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L, a control and a solvent control. Two exposure vessels were established for each concentration and the controls. Following 96 hours of exposure, the control and solvent control averaged 249 and 135 x 10⁴ cells/mL, respectively. Cell density in the 0.0010, 0.010, 0.10, 1.0 and 10 mg/L treatment levels averaged 268, 250, 185, 284 and 285 x 10⁴ cells/mL, respectively. Based on these data, nominal test material concentrations of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 11 - 79 mg a.i./L (95 % CL)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

CELL DENSITY
At 96 hours of exposure, cells exposed to all the treatment levels tested and the controls were observed to be normal. The 96-hour cell density in the control and solvent control averaged 133 and 136 x 10⁴ cells/mL, respectively (pooled control = 134 x 10⁴ cells/mL). Cell densities in the 5.6, 12, 23, 46 and 94 mg a.i./L treatment levels averaged 148, 138, 151, 1.0 and 1.0 x 10⁴ cells/mL, respectively. Statistical analysis (Kruskal-Wallis’ Test), determined no significant reduction in cell density in any treatment level tested as compared to the pooled control.

BIOMASS
The 0- to 72-hour biomass in the control and solvent control averaged 50.6 and 51.5 x 10⁴ cells•days/mL, respectively (pooled control = 51.1 x 10⁴ cells•days/mL). Biomass in the 5.6, 12, 23, 46 and 94 mg a.i./L treatment levels averaged 54.1, 45.3, 55.5, -0.3 and -1.7 cells•days/mL, respectively. Statistical analysis (Kruskal-Wallis' Test) determined no significant difference in biomass in any treatment level tested when compared to the biomass in the pooled control.

GROWTH RATE
The 0- to 72-hour growth rate in the control and solvent control averaged 1.38 and 1.45 days^-1, respectively (pooled control = 1.41 days-1). The 0- to 72-hour growth rate in the 5.6, 12, 23, 46 and 94 mg a.i./L treatment levels averaged 1.39, 1.37, 1.47, -0.05 and -0.40 days^-1, respectively. Statistical analysis (Williams’ Test) determined a significant reduction in the 46 and 94 mg a.i./L treatment levels when compared to the growth rate in the pooled control.

To determine the algistatic/algicidal properties of the test material, an aliquot was removed at test termination from the 100 mg a.i./L nominal solution. The appropriate volume (3.15 mL) of test solution (composite of the three replicates) was diluted with freshly prepared AAP medium to a volume of 50 mL. The resultant concentration of the 50-mL solution was 6.3 mg a.i./L, the lowest nominal concentration tested. The estimated cell density of the 50-mL solution prepared from the 100 mg a.i./L test solution was 0.0365 x 10⁴ cells/mL. The subculture was maintained at a temperature of 23 to 24 °C, a light intensity of 400 to 460 footcandles and a constant shaking rate of 100 rpm for 6 days. After 6 days of exposure, a cell density of 87 x 10⁴ cells/mL was observed in the subculture. This observation indicates that the test material has an algistatic, rather than algicidal effect on the growth of Pseudokirchneriella subcapitata at 100 mg a.i./L (nominal), or a mean measured concentration of 94 mg a.i./L.

Reported statistics and error estimates:

A t-test was conducted to compare the biomass and growth rate of the control to the biomass and growth rate of the solvent control solution. If no significant difference was determined, control and solvent control data were pooled for further statistical analysis to determine treatment level effects. If a significant difference was determined, solvent control data were used for further statistical analyses.
Where required, the EC50 values and their 95 % confidence limits were determined by linear regression of response versus exposure concentration over the range of test concentrations where a clear exposure-response relationship was observed. Four linear regressions were estimated based on (a) untransformed data, (b) untransformed response vs. logarithm-transformed concentration, (c) probit-transformed response vs. untransformed concentration, and (d) probit-transformed response vs. logarithm-transformed concentration. The regression that best fit the data was selected based on the highest coefficient of determination (r²). This regression equation was then applied to estimate the EC values and their 95 % confidence limits, using the method of inverse prediction.
Based on the results of statistical analysis performed for 96-hour cell density and 72-hour total biomass and average growth rate data, the NOEC for p < 0.05 for each parameter when compared to the pooled control data, was determined. The data were first checked for normality using Shapiro-Wilks' Test and for homogeneity of variance using Bartlett's Test. If the data sets passed the test for homogeneity and normality, Williams’ Test was used. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis’ Test was used to determine the NOEC. All statistical determinations were made at the 95 % level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99 % level of certainty was applied.

Analytical Chemistry

Throughout the exposure, the measured concentrations closely approximated the desired nominal concentrations and provided the expected concentration gradient. Mean measured concentrations defined the exposure solutions as 5.6, 12, 23, 46 and 94 mg a.i./L. The 96-hour sample from the 25 mg a.i./L treatment level, without algae present, yielded a measured concentration of 23 mg a.i./L. The equivalent replicate 96-hour test solution with algae present was 22 mg a.i./L, indicating that the presence of algae in the test solution had no impact on the concentration of test material present in the test solution.

Water Quality Parameters

The pH of the test and control solutions ranged from 3.5 to 7.5 at test initiation and was inversely proportional to the test concentration. At 96 hours of exposure, the pH of the test and control solutions ranged from 3.5 to 9.8. Conductivity of the exposure and control solutions measured at test initiation and termination ranged from 80 to 170 μmhos/cm. Continuous temperature monitoring established that the temperature was maintained at 23 to 24 °C throughout the study period. Light intensity of the test area ranged from 300 to 420 footcandles (3200 to 4500 lux) throughout the exposure period. The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 35 to 61 μE/m²/s. The shaking rate was maintained at a constant rate of 100 rpm throughout the exposure.

Table 1: Mean Growth Rates After 24, 48 and 72 Hours of Exposure to the Test Material

Mean measured conc. (mg a.i./L)	Observation interval (hours)			Percent inhibition*
	0 - 24	0 - 48	0 - 72
Control	1.94	1.46	1.38	NA
Solvent control	1.50	1.49	1.45	NA
5.6	1.71	1.56	1.39	1
12	1.80	1.41	1.37	3
23	1.68	1.50	1.47	-4
46	-0.26	-0.12	-0.05**	104
94	-1.05	-0.57	-0.40**	128

*Percent inhibition relative to the pooled control (0 - 72 hours growth rate 1.45)

**Significantly reduced compared to pooled control, based on Williams’ Test

Validity criteria fulfilled:

yes

Conclusions:

In terms of growth rate, the 72 hour NOEC was determined to be 23 mg a.i./L. The 72-hour ErC50 for growth rate was calculated to be 30 mg a.i./L, with 95 % confidence limits of 11 to 79 mg a.i./L.

Executive summary:

The toxicity of the test material to the freshwater alga, Pseudokirchneriella subcapitata, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 123-2, OECD 201 and EU Method C.3.

Based on the results of a preliminary range-finding exposure, nominal dose levels of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent control solution was prepared with AAP medium containing 0.10 mL/L DMF. Additional untreated AAP medium was used to prepare the control. Three replicates were prepared for each concentration of test material, solvent and untreated control. Approximately 35 minutes after the test solutions were added to the test flasks (100 mL per flask), a 0.392-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 255 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10⁴ cells/mL.

At each 24-hour interval, cell counts were conducted on the three replicate vessels (A, B and C) of the treatment and control vessels using a haemocytometer and compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were also made and recorded at each 24-hour interval.

Statistical analysis (Kruskal-Wallis’ Test), determined no significant reduction in cell density in any treatment level tested as compared to the pooled control. Therefore, the NOEC was empirically estimated to be 23 mg a.i./L, the highest concentration tested with < 10 % inhibition of cell density. The 96-hour EC50 for cell density was calculated to be 32 mg a.i./L, with 95 % confidence limits of 9.4 to 110 mg a.i./L.

Statistical analysis (Kruskal-Wallis' Test) determined no significant difference in biomass in any treatment level tested when compared to the biomass in the pooled control. Therefore, the NOEC was empirically estimated to be the highest concentration tested which exhibited < 10 % inhibition, 23 mg a.i./L. The 72-hour EbC50 for biomass was calculated to be 32 mg a.i./L, with 95 % confidence limits of 7.6 to 130 mg a.i./L.

Statistical analysis (Williams’ Test) determined a significant reduction in the 46 and 94 mg a.i./L treatment levels when compared to the growth rate in the pooled control. The NOEC was determined to be 23 mg a.i./L. The 72-hour ErC50 for growth rate was calculated to be 30 mg a.i./L, with 95 % confidence limits of 11 to 79 mg a.i./L.

Investigation of the algistatic/algicidal properties of the test material revealed the test material has an algistatic, rather than algicidal effect on the growth of Pseudokirchneriella subcapitata at 100 mg a.i./L (nominal), or a mean measured concentration of 94 mg a.i./L.

Description of key information

FRESHWATER
72 hr ErC50 = 30 mg/L; 72 h NOEC = 23 mg/L (Pseudokirchneriella subcapitata), EPA OPP 123-2, OECD 201 and EU Method C.3, Hoberg (2003)
120 hr ErC50 = 23.3 - 47.2 mg/L (empirically determined); 120 hr NOEC (growth rate) = 23.3 mg/L; 120 hr EbC50 = 27.8 mg/L; 120 hr NOEC (biomass) = 23.3 mg/L (Anabaena flos-aquae), EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), OECD 201 and EU Method C.3, Hancock et al. (2007)
72 hr ErC50 = 21 mg/L; 72 hr NOEC = 23 mg/L (Navicula pelliculosa) EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3, Hoberg (2002)
SALTWATER
72 hr ErC50 = > 100 mg/L; 72 hr NOEC = 13 mg/L (Skeletonema costatum), EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3, Hoberg (2002)

Key value for chemical safety assessment

EC50 for freshwater algae:

21 mg/L

EC50 for marine water algae:

100 mg/L

EC10 or NOEC for freshwater algae:

23 mg/L

EC10 or NOEC for marine water algae:

13 mg/L

Additional information

Freshwater

Four studies investigating toxicity of the substance to aquatic algae and cyanobacteria are available. All four studies were conducted under GLP conditions and in accordance with standardised guidelines; three were subsequently assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997). the study by Hoberg (2002) conducted with Anabaena flos-aquae was assigned a reliability score of 3 and the study disregarded on the basis that low growth, as well as variability in growth, during the first 96 hours of the test resulted in the data not being reliable.

In the study reported by Hoberg (2003) the toxicity of the test material to the freshwater alga, Pseudokirchneriella subcapitata, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 123-2, OECD 201 and EU Method C.3.

Based on the results of a preliminary range-finding exposure, nominal dose levels of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent control solution was prepared with AAP medium containing 0.10 mL/L DMF. Additional untreated AAP medium was used to prepare the control. Three replicates were prepared for each concentration of test material, solvent and untreated control. Approximately 35 minutes after the test solutions were added to the test flasks (100 mL per flask), a 0.392-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 255 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10⁴ cells/mL.

At each 24-hour interval, cell counts were conducted on the three replicate vessels (A, B and C) of the treatment and control vessels using a haemocytometer and compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were also made and recorded at each 24-hour interval.

Statistical analysis (Kruskal-Wallis’ Test), determined no significant reduction in cell density in any treatment level tested as compared to the pooled control. Therefore, the NOEC was empirically estimated to be 23 mg a.i./L, the highest concentration tested with < 10 % inhibition of cell density. The 96-hour EC50 for cell density was calculated to be 32 mg a.i./L, with 95 % confidence limits of 9.4 to 110 mg a.i./L.

Statistical analysis (Kruskal-Wallis' Test) determined no significant difference in biomass in any treatment level tested when compared to the biomass in the pooled control. Therefore, the NOEC was empirically estimated to be the highest concentration tested which exhibited < 10 % inhibition, 23 mg a.i./L. The 72-hour EbC50 for biomass was calculated to be 32 mg a.i./L, with 95 % confidence limits of 7.6 to 130 mg a.i./L.

Statistical analysis (Williams’ Test) determined a significant reduction in the 46 and 94 mg a.i./L treatment levels when compared to the growth rate in the pooled control. The NOEC was determined to be 23 mg a.i./L. The 72-hour ErC50 for growth rate was calculated to be 30 mg a.i./L, with 95 % confidence limits of 11 to 79 mg a.i./L.

Investigation of the algistatic/algicidal properties of the test material revealed the test material has an algistatic, rather than algicidal effect on the growth of Pseudokirchneriella subcapitata at 100 mg a.i./L (nominal), or a mean measured concentration of 94 mg a.i./L.

  

In the study reported by Hancock et al. (2007) the toxicity of the test material to the freshwater alga, Anabaena flos-aquae, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II), OECD 201 and EU Method C.3.

The algae were exposed to nominal concentrations of test material of 0 (medium control), 3.13, 6.25, 12.5, 25.0, 50.0 and 100 mg/L, under static conditions, for a period of 120 hours. Three replicates at each test level and six replicates at the medium control level were inoculated with approximately 20 000 cells/mL. The additional replicate at each test and control level was not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the interference of the test material and to monitor pH and concentration of the test material without the algal biomass.

At approximately 24, 48, 72, 96, and 120 hours, samples were withdrawn from each replicate for determination of particle counts and cell volume. Each replicate sample was counted three times (for a total of six counts per replicate per day). At test termination, a microscopic visual observation on the general health of the cells at each test level will be made.

Mean cell volumes at 120 hours were 7964, 7513, 8184, 8118, 7048, 19.0, and 0.290 x 10^4 μm³/mL for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L test levels, respectively. Response relative to the controls ranged from 3 % stimulation to 100 % inhibition of cell volume. Mean biomass values at 120 hours were 235964, 213444, 234988, 245040, 202260, -1577, and -3257 for the control, 3.10, 5.62, 11.6, 23.3, 47.2, and 94.7 mg test material/L/L test levels, respectively. Response relative to the controls ranged from 4 % stimulation to 101 % inhibition of biomass.

Under the conditions of the study, the 120-hour ErC50 was empirically determined to be between 23.3 and 47.2 mg a.i./L with a NOEC value of 23.3 mg a.i./L. In terms of biomass, the 120 hour EbC50 was determined to be 27.8 mg a.i./L, the corresponding NOEC was 23.3 mg a.i./L.

 

In the study reported by Hoberg (2002) the toxicity of the test material to the freshwater alga, Navicula pelliculosa, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3.

Based on the results of preliminary testing, nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L were selected for the definitive exposure. A solvent control was also prepared by diluting DMF in AAP medium. Additional untreated AAP medium was used for the control. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Within 45 minutes of preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 0.263 mL of an inoculum of Navicula pelliculosa cells, at a density of 380 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell density of approximately 1.0 x 10⁴ cells/mL.

At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval. At test termination, due to the habit of Navicula pelliculosa cells to clump, the solutions were vigorously pipetted multiple times to break up clumped cells and achieve a homogeneous suspension prior to removing a sample for cell counts.

Temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period.

Prior to the test termination observation interval, the test solutions were vigorously pipetted multiple times to disperse clumped cells and provide a homogeneous suspension for counting. At test termination (120 hours), cells exposed to the treatment levels tested and the controls were observed to be normal.

Under the conditions of the study a significant reduction in both cell density, and biomass, was observed in the 12, 23, 48 and 100 mg a.i./L treatment levels as compared to the pooled control data. In addition, statistical analysis determined a significant reduction in treatment levels 48 and 100 mg a.i./L when compared to the growth rate of the solvent control.

Based on cell density, the 120-hour NOEC was determined to be 6.0 mg a.i./L. The 120-hour EC25 and EC50 value (corresponding 95 % confidence intervals) were calculated to be 11 mg a.i./L (2.6 to 38 mg a.i./L) and 22 mg a.i./L (6.0 to 81 mg a.i./L), respectively. Based on cell biomass, the NOEC was determined to be 6.0 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 18 mg a.i./L (5.4 to 59 mg a.i./L). Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 23 mg a.i./L. The 72-hour ErC50 (corresponding 95 % confidence intervals) was calculated to be 21 mg a.i./L (3.7 to 140 mg a.i./L), NOEC = 23 mg a.i./L.

In the disregarded study reported by Hoberg (2002), the toxicity of the test material to the freshwater alga, Anabaena flos-aquae, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3.

Based on the results of preliminary testing, nominal concentrations of 0.40, 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L were selected for the definitive exposure. A solvent control solution was prepared with AAP medium containing 0.10 mL/L DMF. Additional untreated AAP medium was used to prepare the control. Three replicates were prepared for each concentration of test material, solvent and untreated control. Approximately one hour after preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 1.6 mL of an inoculum of Anabaena flos-aquae cells, at a density of 62 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell density of approximately 1.0 x 10⁴ cells/mL.

At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval.

Due to the habit of Anabaena flos-aquae cells to grow in filaments, at test termination the solutions were vigorously pipetted multiple times to break up the filaments and achieve a homogeneous suspension prior to removing a sample for cell counts.

Temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period.

At test termination (120 hours), cells exposed to the 38 mg a.i./L treatment level were observed to be bloated. Cells exposed to the remaining treatment levels (0.39, 1.0, 2.5, 6.2, and 16 mg a.i./L) tested and the controls were observed to be normal. No cells were observed in the 100 mg a.i./L treatment level. The 120-hour cell density in the control and solvent control culture both averaged 63 x 10⁴ cells/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control cell density; therefore control and solvent control data were pooled (pooled control = 63 x 10⁴ cells/mL). Cell density in the 0.39, 1.0, 2.5, 6.2, 16, 38 and 100 mg a.i./L treatment levels averaged 67, 73, 63, 64, 59, 13 and 0 x 10⁴ cells/mL, respectively. Based on the results of Sharpiro-Wilks’ Test and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. A significant reduction in cell density was observed in the 38 and 100 mg a.i./L treatment levels as compared to the pooled control. The 120-hour NOEC was determined to be 16 mg a.i./L. The 120-hour EC25 and EC50 value (corresponding 95 % confidence intervals) was calculated to be 15 mg a.i./L (5.0 to 40 mg a.i./L) and 27 mg a.i./L (9.9 to 76 mg a.i./L), respectively. The 24, 48, 72 and 96 hour EC25 and EC50 values were not calculated because a well-defined concentration-response was not observed. The lack of a well-defined response was primarily due to difficulty in homogeneous dispersion of cells prior to test termination. Anabaena flos-aquae is filamentous and cells were dispersed at test termination by rapid pipetting of the solution.

The total biomass in the control and solvent control averaged 16.9 and 12.2 x 10⁴ cells·days/mL, respectively. Statistical analysis (t-Test) determined no significant difference between control and solvent control biomass; therefore control and solvent control data were pooled (pooled control = 14.6 x 10⁴ cells·days/mL). Total biomass in the 0.39, 1.0, 2.5, 6.2, 16, 38 and 100 mg a.i./L treatment levels averaged 7.7, 13.9, 4.2, 5.0, 8.3, 6.1, -0.8 cells·days/mL, respectively. The EC50 and NOEC values were not determined since a well defined concentration-response was not observed, as previously discussed. Similarly, growth rates were not presented due to the inability to determine precise cell counts.

 

Saltwater

In the study reported by Hoberg (2002) the toxicity of the test material to the marine diatom, Skeletonema costatum, was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 122 -2 and 123-2, OECD 201 and EU Method C.3.

During the study, the organisms were exposed to nominal concentrations of test material of 6.3, 13, 25, 50 and 100 mg a.i./L, under static conditions, for 120 hours. A solvent (DMF) control and untreated control (artificially enriched seawater) were included. Three replicates were prepared for each concentration of test material, solvent and untreated control.

Approximately one hour after preparation of the test solutions and addition to the exposure flasks (100 mL/flask), 0.938 mL of an inoculum of Skeletonema costatum cells, at a density of 107 x 10⁴ cells/mL, was aseptically introduced into each flask. This inoculum provided the required cell density of approximately 1.0 x 10⁴ cells/mL.

At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment level and controls using a haemocytometer and a compound microscope. One sample was removed from each flask for counting. One or more haemocytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval. Furthermore, temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 120-hour exposure period.

Under the conditions of the test, the findings for cell density showed all treatments to be similar to the pooled control data, therefore the 120-hour NOEC was determined to be 100 mg a.i./L. The 120-hour EC25 and EC50 values were empirically estimated to be > 100 mg a.i./L, the highest mean measured concentration tested. Statistical analysis of the cell biomass data determined a significant difference in biomass in the 25, 50 and 100 mg a.i./L treatment levels when compared to the biomass in the pooled control (20.9 x 10⁴ cells·days/mL). Based on these results, the 72-hour NOEC for total biomass was determined to be 13 mg a.i./L. The 72-hour EbC50 (corresponding 95 % confidence intervals) was calculated to be 77 mg a.i./L (13 to 1000 mg a.i./L). Statistical analysis of the growth data determined a significant reduction in treatment levels 25, 50 and 100 mg a.i./L when compared to the growth rate of the pooled control. Based on Williams’ Test, the 72-hour NOEC for growth rate was determined to be 13 mg a.i./L. The 72-hour ErC50 was empirically estimated to be >100 mg a.i./L, the highest mean measured concentration tested.