Clorofene

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:

key study

Study period:

16 to 20 March 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only four replicates for the control

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OCSPP 850.4500

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.0043, 0.011, 0.027, 0.067, 0.17 and 0.50 mg a.i./L
- Sampling method: The concentration of the test item was measured in test solution samples at test start (0 h) and test end (96 h). The 0-hour samples were collected from parent solutions. The 96-hour biotic samples were collected after compositing replicate A, B, C, and D solutions (control and all test solutions) by treatment.
- Sample storage conditions before analysis: All solutions were stored refrigerated while not in use.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.010 mg a.i./mL primary standard was prepared by transferring 0.0101 g of the test item to a 1.000 mL glass volumetric flask, and bringing the flask to volume with test medium. The primary standard was sonicated for approx 90 min and then stirred using a teflon stir bar for approx 2 h. Appropriate aliquots of the primary standard solution were diluted in 0.5 or 1.0 L of test medium to prepare the required test item concentrations.
- Controls: yes, medium only

Test organisms (species):

Navicula pelliculosa

Details on test organisms:

TEST ORGANISM
- Common name: Diatom
- Source: University of Texas at Austin (UTEX)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock.

ACCLIMATION
- Acclimation period: 72 h
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: No physical abnormalities were observed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23.1 - 24.6 °C (Control)
22.9 - 24.7 °C (test item concentrations)

pH:

7.5 - 8.3 (Control)
7.5 - 8.2 (test item concentrations)

Nominal and measured concentrations:

Control, 0.0043. 0.011, 0.027. 0.067. 0.17. and 0.50 mg a.i./L (nominal)
Control, 0.00457, 0.00990, 0.0218, 0.0569, 0.153 and 0.471 mg a.i./L (geometric mean measured concentration)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (with foam stoppers)
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks, 100 mL of the appropriate parent solution and 150 mL headspace
- Initial cells density: 1.0 x 10E+04 cells/mL
- Control end cells density: 121 x 10E+04 cells/mL (after 96 h)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Others: Abiotic replicate of the nominal 0.0043 mg a.i./L test substance treatment was not inoculated with algae

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium + Si)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshwater algal nutrient medium (FWAM + Si correspond to AAP medium) prepared by the addition of reagent water which is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, demineralization, and organic adsorption cartridges, and then through a 0.2 µm filter.
- Analysis: Nitrogen, Nitrate: 0.304 mg/L; Phosphorus, Total: <0.05 mg/L; Nitrogen, Nitrite: <0.05 mg/L;
- Metals: Barium 0.0194 mg/L; Boron 0.385 mg/L; Calcium 61.6 mg/L; Iron: 0.162 mg/L; Magnesium: 28 mg/L; Potassium: 7.56 mg/L; Sodium: 28.4 mg/L; Zinc: 0.0313 mg/L
- Pesticides: all tested chlorinated pesticides were below the reporting limit (1.25 µg/L).
- Polychlorinated Biphenyls (PCBS): below detection limit (1 µg/L)
- Chlorinated Herbicides: below detection limit (0.2 µg/L)
- Organophosphorous Pesticides: below detection limit (0.002 mg/L)
- Culture medium different from test medium: no
- Intervals of water quality measurement: test start (0 h) and test end (96 h)

OTHER TEST CONDITIONS
- Adjustment of pH: 7.5 ± 0.1 using 0.1 N NaOH and 0.1 N HCl
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: 3.754 to 4.698 lux.
- Others: the flasks were randomly positioned daily using a computer-generated random number table and incubated in a temperature controlled environmental chamber; flasks were swirled by hand on a daily basis.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: direct microscopic counting with a hemacytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.9
Range finding study
- Test concentrations: control, 0.0010. 0.010, 0.10, 1.0. and 10 mg a.i./L
- Results used to determine the conditions for the definitive study: Based on these results, a nominal concentration range of control, 0.0043, 0.011. 0.027, 0.067, 0.17. and 0.50 mg a.i./L was selected for the definitive test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.0303-0.0383 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.155 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.148-0.162 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.0503-0.0707 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.435 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.414-0.456 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.021 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.0162-0.0252 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.101 - 0.119 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.0236- 0.0309 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.115 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.109 - 0.121 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.059 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.0477 - 0.0695 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

> 0.471 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

not statistically sound

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.0242 - 0.0357 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.102 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.0945 - 0.109 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no

Reported statistics and error estimates:

All statistical analyses were performed with SAS software (Version 9.3 for Windows). All statistical analy ses were conducted compared to the control.
The NOEC values, based on area under the growth curve, growth rate, and yield, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett's test, a Shapiro-Wilk's test and a Levene's test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk's and Levene's tests indicated normality and insignificant heterogeneity (i.e., p >0.01), the analysis was performed on the non- transformed raw data. In instances of non-normality or heterogeneity (i.e., p <0.01), a square root transformation was performed.

Table 1: Measured concentrations of the test item in test solutions during the 96-h growth inhibition test w ith the freshwater diatom, Navicula pelliculosa

Nominal Concentration	Measured Concentration as mg a.i./L (Percent Nominal)
(mg a.i./L)	0 h		96 h	Mean measured concentration (mg a.i./L)	Geometric mean measured concentration (mg a.i./L)
Control	<MQLa		<MQLa	<MQLa	<MQLa
0.0043	0.00408 (95)		0.00512 (119)c	0.00460 (107)	0.00457(106)
0.0043 Abiotic	0.00408 (95)		0.00562 (131)c	0.00485 (113)	0.00479(111)
0.011	0.0115(105)		0.00853 (78)	0.0100 (91)	0.00990 (90)
0.027	0.0255 (94)		0.0186 (69)	0.0221 (82)	0.0218 (81)
0.067	0.0681 (102)		0.0476(71)	0.0579(86)	0.0569 (85)
0.17	0.169 (99)		0.138 (81)	0.154 (91)	0.153 (90)
0.50	0.506(101)		0.439 (88)	0.473 (95)	0.471 (94)
QC Fortification Spikes (% Recovery)
Low Spike (0.00403)	0.00354 (88)	0.00553 (137)		--	--
High spike (0.600)	0.608 (101)	0.649 (108)		--	--

a: MQL = 0.00372 mg a.i./L; b: The reported value for Level 1 abiotic hour 0 is the result from Level 1 hour 0 as the samples were taken from parent solutions.

c: Sample re-analyzed in duplicate, average of duplicate re-analyses reported

Table 2: Cell density values for Navicula pelliculosa during a 96 -h exposure to the test item

Geometric Mean Measured Concentration (mg a.i./L)	Mean Cell Density (cells/mL x 104)
	24 h	48 h	72 h	96 h
Control	3.14	9.87	40.5	121
0.00457	3.00	10.2	38.6	120
0.00990	3.42	9.97	38.8	125
0.0218	3.44	10.7	38.7	113
0.0569	3.31	10.0	27.8	94.3
0.153	1.95	7.61	17.0	36.7
0.471	1.53	3.14	5.92	12.3

 

Table 3: Growth rate values from time zero for Navicula pelliculosa during a 96 -h exposure to the test item

Geometric Mean Measured Concentration (mg a.i./L)	Mean Growth Rate (cells/mL/hour)				% Inhibition
	0-24 h	0-48 h	0-72 h (%CV)	0-96 h (%CV)	72 h	96 h
Control	0.0469	0.0475	0.0513 (CV: 0)	0.0498 (CV: 1)	—	—
0.00457	0.0452	0.0481	0.0506 (CV: 1)	0.0498 (CV: 1)	1	0
0.00990	0.0505	0.0477	0.0507 (CV: 1)	0.0502 (CV: 1)	1	-1
0.0218	0.0509	0.0490	0.0506 (CV: 2)	0.0491 (CV: 1)	1	1
0.0569	0.0492	0.0478	0.0460* (CV: 3)	0.0473* (CV: 1)	10	5
0.153	0.0269*	0.0420*	0.0392* (CV: 3)	0.0374* (CV: 3)	24	25
0.471	0.0168*	0.0235*	0.0245* (CV: 7)	0.0260* (CV: 2)	52	48

* Significant reduction in growth rate as compared to the control (Dunnett's test, p = 0.05).

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	121	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	15.8	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	4,04	Yes

 

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 to 20 March 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only four replicates for the control

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OCSPP 850.4550

Version / remarks:

2012

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.0297, 0.0677, 0.179. 0.410, 1.15. and 2.91 mg a.i./L
- Sampling method: The concentration of the test item was measured in test solution samples at test start (0 h) and test end (96 h). The 0-hour samples were collected from parent solutions. The 96 h biotic samples were collected after compositing replicate A, B, C, and D solutions (control and all test solutions) by treatment.
- Sample storage conditions before analysis: All solutions were stored refrigerated while not in use.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 0.010 mg a.i./mL primary standard was prepared by transferring 0.0101 g of test substance to a 1000 mL glass volumetric flask, and bringing the flask to volume with test medium. The primary standard was sonicated for approx 90 min and then stirred for approx 2 h at 25 °C. Appropriate aliquots of the primary standard solution were diluted to achieve the required test item concentrations.
- Controls: medium only

Test organisms (species):

Anabaena flos-aquae

Details on test organisms:

TEST ORGANISM
- Common name: Blue algae
- Source: University of Texas at Austin (UTEX), USA
- Age of inoculum (at test initiation): Algal culture used for this test was three days old at test initiation.
- Method of cultivation: Cultures were maintained in a temperature-controlled environmental chamber under continuous light.

ACCLIMATION
- Acclimation period: Periodically, new cultures were cloned from an existing culture derived from the parent stock.
- Culturing media and conditions: same
- Any deformed or abnormal cells observed: no deformation was observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

after 96 h of exposure a recovery period was followed up

Test temperature:

24.3 - 25.3 °C (Control)
24.2 - 25.3 °C (Test item concetrations)

pH:

7.5 - 7.7 (Control)
7.4 - 8.0 (Test item concentrations)

Nominal and measured concentrations:

0 (control), 0.031, 0.077. 0.19, 0.48, 1.2, and 3.0 mg a.i./L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (with foam stoppers)
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks, 100 mL of the appropriate parent solution and 150 mL headspace
- Initial cells density: 1.0 x 10E+04 cells/mL
- Control end cells density: 51.1 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Others: Abiotic replicate of the nominal 0.0031 mg a.i./L test substance treatment was not inoculated with algae

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: freshwater algal nutrient medium (FWAM correspond to AAP medium) prepared by the addition of reagent water which is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, de-mineralization, and organic adsorption cartridges, and then through a 0.2-µm filter.
- Analysis: Nitrogen, Nitrate: 0.304 mg/L; Phosphorus, Total: <0.05 mg/L; Nitrogen, Nitrite: <0.05 mg/L;
- Metals: Barium 0.0194 mg/L; Boron 0.385 mg/L; Calcium 61.6 mg/L; Iron: 0.162 mg/L; Magnesium: 28 mg/L; Potassium: 7.56 mg/L; Sodium: 28.4 mg/L; Zinc: 0.0313 mg/L
- Pesticides: all tested chlorinated pesticides were below the reporting limit (1.25 µg/L).
- Polychlorinated Biphenyls (PCBS): below detection limit (1 µg/L)
- Chlorinated Herbicides: below detection limit (0.2 µg/L)
- Organophosphorous Pesticides: below detection limit (0.002 mg/L)
- Culture medium different from test medium: no
- Intervals of water quality measurement: test start (0 h) and test end (96 h)

OTHER TEST CONDITIONS
- Adjustment of pH: 7.5 ± 0.1 using 0.1 N NaOH and 0.1 N HCl
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: 2.261 to 2.284 lux
- Others: the flasks were randomly positioned daily using a computer-generated random number table and incubated in a temperature controlled environmental chamber; flasks were swirled by hand on a daily basis.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: direct microscopic counting with a hemacytometer

Recovery:
- Concentrations: At test end the control and all test concentrations exhibiting a maximal inhibition of healthy cell counts relative to the control were selected for the recovery test.
- Conditions: 2 mL samples were taken and diluted to 100 mL with untreated test medium. The dilution was sufficient to result in a test item concentration that would not inhibit algal growth and growth rate based on visual observations during termination of the definitive test. A single recovery replicate was prepared for the control and 2.77 mg a.i./L geometric mean measured treatment. The recovery exposure replicates were incubated under the same general conditions used during the definitive exposure. Recovery replicates were monitored for growth on day 6 of the recovery exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
Range finding study
- Test concentrations: control, 0.0010, 0.010, 0.10. 1.0, and 10 mg a.i./L
- Results used to determine the conditions for the definitive study: Based on these results, a nominal concentration range of Control, 0.031, 0.077, 0.19, 0.48, 1.2, and 3.0 mg a.i./L was selected for the definitive test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.217 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.199 - 0.236 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.563 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.543 - 0.584 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.159 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.794 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.779 - 0.808 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.989 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.971 - 1.01 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.159 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

IC10

Effect conc.:

0.199 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.183 - 0.215 mg a.i./L

Duration:

72 h

Dose descriptor:

IC50

Effect conc.:

0.474 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.481 - 0.544 mg a.i./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.159 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.153 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.130 - 0.176 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.513 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% Confidence Limits

Remarks:

0.481-0.544 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.061 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.885 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits

Remarks:

0 - 8.30 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

1.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Limits

Remarks:

0 - 6.39 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Remarks:

Statistically significant at 0.159 mg a.i./L (Dunnett's 1-tailed t test, a = 0.05), but not found to be biologically significant

Effect conc.:

0.159 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.089 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidential limits

Remarks:

0.0503 - 0.127 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.456 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% Confidential limits

Remarks:

0.379 - 0.532 mg a.i./L

Details on results:

- Exponential growth in the control (for algal test): yes
- Other: Recovery was assessed for the 2.77 mg a.i./L geometric mean measured treatment utilizing the same growth chamber as the definitive test. All environmental parameters for the recovery test were within expected ranges. Although not measured directly, lighting was set at normal study conditions of 2.150 ± 320 lux for the duration of the recovery test. Data from the electronic data logger confirmed that the temperature of the incubator remained within the 24 ± 2 °C range specified in the protocol throughout the recovery test. Based on the 376 x increase in cell count in 6 days, the effects upon growth of Anabaena flos-aquae were found to be algistatic at a geometric mean measured test item concentration of 2.77 mg a.i./L.

Reported statistics and error estimates:

All statistical analyses were performed with SAS software (Version 9.3 for Windows). All statistical analy ses were conducted compared to the control.
The NOEC values, based on area under the growth curve, growth rate, and yield, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett's test, a Shapiro-Wilk's test and a Levene's test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk's and Levene's tests indicated normality and insignificant heterogeneity (i.e., p >0.01), the analysis was performed on the non- transformed raw data. In instances of non-normality or heterogeneity (i.e., p <0.01), a square root transformation was performed.

Analytical Results:

Measured concentrations of the test item at test start ranged between 85 to 97% and at test end between 71 to 88% of the nominal concentrations. Due to the fact that the measured test concentration were below 80 % of nominal at test end the toxicological endpoints were evaluated using the actual measured concentrations based on the geometric mean of each test item concentration.

Table 1: Analytical Results: Measured concentrations of the test item in test solutions during the 96 -h growth inhibition test with the cyanobacterium, Anabaena flos-aquae

Nominal Concentration	Measured Concentration as mg a.i./L (percent nominal)
(mg a.i./L)	0 h	96h	Mean measured Concentration (mg a.i./L)	Geometric Mean measured Concentration (mg a.i./L)
0 (Control)	<MQLa	<MQLa	<MQLa	<MQLa
0.031	0.0297 (96)	0.0233 (75)	0.0265 (85)	0.0263 (85)
0.031 (abiotic)	0.0297 (96)	0.0306 (99)	0.0302 (97)	0.0301 (97)
0.077	0.0677 (88)	0.0546 (71)	0.0612 (79)	0.0608 (79)
0.19	0.179 (94)	0.142 (75)	0.161 (85)	0.159 (84)
0.48	0.410 (85)	0.344 (72)	0.377 (79)	0.376 (78)
1.2	1.15 (96)	0.904 (75)	1.03 (86)	1.02 (85)
3.0	2.91 (97)	2.63 (88)	2.77 (92)	2.77 (92)

Biological Results:

Table 2: Cell Density values for Anabaena flos-aquae during a 96-h exposure to the test item

Geometric Mean Measured Concentration	Mean Cell Density (cells/mL x 10E+04)
(mg a.i/L)	24 h	48 h	72h	96 h
0 (Control)	2.42	7.03	21.3	51.5
0.0263	2.53	7.20	21.3	49.4
0.0608	2.39	7.14	22.1	42.8
0.156	2.39	6.86	19.9	36.7
0.376	2.28	5.86	14.1	29.5
1.02	2.11	2.06	3.58	16.8
2.77	1.75	0.750	0.861	0.639

Target inoculation cell density was 1.0 x 10⁴cells/mL

Table 3: Growth rate values from test start for Anabaena flos-aquae during a 96-h exposure to the test item

Geometric Mean Measured Concentration (mg a.i./L)	Mean Growth Rate (cells/mL/h)				% Inhibition
	0-24 h	0-48 h	0-72 h (%CV)	0-96 h (%CV)	72 h	96 h
Control	0.0384	0.0415	0.0430 (CV: 3)	0.0414 (CV: 2)	—	—
0.0263	0.0402	0.0419	0.0430 (CV: 3)	0.0410 (CV: 2)	0	1
0.0608	0.0378	0.0418	0.0435 (CV: 2)	0.0395 (CV: 2)	-1	5
0.159	0.0379	0.0409	0.0421 (CV: 3)	0.0379a (CV: 2)	2	8
0.376	0.0359	0.0376*	0.0373* (CV: 2)	0.0357* (CV: 2)	14	14
1.02	0.0328*	0.0158*	0.0182* (CV: 9)	0.0298* (CV: 2)	58	28
2.77	0.0250*	-0.00543*	-0.00176* (CV: -171)	-0.00502* (CV: -95)	104	112

* Significant reduction in growth rate as compared to the control (Dunnett's test, p = 0.05).

a Statistically significant at 0.159 mg a.i./L (Dunnett's 1-tailed t test, a = 0.05), but not found to be biologically significant.

Table 4: Validity criteria according OECD 201

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	21.35	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	13.5	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	2.12	Yes

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 - 16 June 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only four replicates for the control

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OCSPP 850.4500

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test item concentrations and control
- Sampling method: samples were taken at test start (0 h) and test end (96 h); 0 h samples were collected from parent solutions. 96 h samples were collected after combining replicate solutions by treatment.
- Sample storage conditions before analysis: solutions were stored refrigerated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 0.010 mg a.i./mL primary standard was prepared by transferring 0.0101 g of the test item to a 1.000 mL glass volumetric flask, and bringing the flask to volume with test medium. The primary standard was sonicated for approx 90 min and then stirred using a teflon stir bar for approx 2 h. Appropriate aliquots of the primary standard solution were diluted in 0.5 or 1.0 L of test medium to prepare the required test item concentrations.
- Controls: yes, medium only

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Common name: marine diatom
- Source: University of Texas at Austin (UTEX)
- Age of inoculum (at test initiation): culture used for this test was three days old at test initiation
- Method of cultivation: Periodically, new Skeleionema cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing.

ACCLIMATION
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: No physical abnormalities were observed.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

21.7 - 21.9 °C (Control)
21.7 - 22.0 °C (test item concentrations)

pH:

8.0 (Control)
7.5 - 8.1 (test item concetrations)

Salinity:

30.9 to 31.0 ‰

Nominal and measured concentrations:

Control, 0.0051, 0.013, 0.032. 0.080, 0.20, and 0.50 mg a.i./L (nominal)
Control, 0.00630, 0.0113, 0.0283, 0.0655, 0.160 and 0.433 mg a.i./L (based on the geometric mean measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with with foam stoppers)
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, 100 mL of the appropriate parent solution and 150 mL headspace
- Initial cells density: 1.0 x 10E+04 cells/mL
- Control end cells density: 63 x 10E+04 cells/mL (after 96 h)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Others: Abiotic replicate of the nominal 0.0051 mg a.i./L test substance treatment was not inoculated with algae

GROWTH MEDIUM
- Detailed composition if non-standard medium was used: The medium was prepared by the addition of appropriate reagent grade salts to filtered synthetic seawater. The synthetic seawater was prepared by adding a commercial salt mix (Marinemix manufactured by Wiegandt GmbH) to autoclaved ABC reagent water until salinity reached approx 30 ± 2 ‰. ABC reagent water is produced by passing reverse-osmosis water through a series of deionization tanks, a laboratory water purification system consisting of carbon, demineralization, and organic adsorption cartridges, and then through a 0.2 µm filter. The synthetic seawater was then passed through 0.2 µm Millipore® filters for definitive testing.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ABC Salt Water
- Analysis: Nitrogen, Nitrate: 0.148 mg/L; Phosphorus, Total: <0.05 mg/L; Nitrogen, Nitrite: <0.05 mg/L;
- Metals: Barium 0.0157 mg/L; Boron 3.05 mg/L; Calcium 218 mg/L; Iron: < 0.02 mg/L; Magnesium: 643 mg/L; Potassium: 210 mg/L; Sodium: 5720 mg/L; Zinc: <0.01 mg/L
- Pesticides: all tested chlorinated pesticides were below the reporting limit (1.25 µg/L).
- Polychlorinated Biphenyls (PCBS): below detection limit (1 µg/L)
- Chlorinated Herbicides: below detection limit (0.2 µg/L)
- Organophosphorous Pesticides: below detection limit (0.002 mg/L)
- Culture medium different from test medium: no
- Intervals of water quality measurement: test start (0 h) and test end (96 h)

OTHER TEST CONDITIONS
- Adjustment of pH: medium was pH-adjusted to 8.0 ± 0.1 using 0.1 N NaOH/HCl
- Photoperiod: 14 h light: 10 h dark
- Light intensity and quality: 4.513 to 4,737 lux
- Salinity (for marine algae): 30.9 to 31.0 ‰
- Others: the flasks were randomly positioned daily using a computer-generated random number table and incubated in a temperature controlled environmental chamber; flasks were swirled by hand on a daily basis.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: microscopic counting with a hemacytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
Range finding study
- Test concentrations: Control, 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L
- Results used to determine the conditions for the definitive study: The percent change in cell density at 96 h, as compared to the control mean cell density, was -2. 2, 52. 97 and 98% in the 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L treatments, respectively. Based on these results, a nominal concentration range of Control, 0.0051, 0.013, 0.032, 0.080, 0.20 and 0.50 mg a.i./L was selected for the definitive test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.00811 and 0.0138 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.056 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% confidential limits

Remarks:

0.0503 and 0.0614 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.045 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.0385 and 0.0518 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.155 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential limits

Remarks:

0.146 and 0.163 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

IC10

Effect conc.:

0.009 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.00616 and 0.0128 mg a.i./L

Duration:

96 h

Dose descriptor:

IC50

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Remarks on result:

other: 95% confidential limits

Remarks:

0.0395 and 0.0518 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.006 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: no

Reported statistics and error estimates:

All statistical analyses were performed with SAS software (Version 9.3 for Windows). All statistical analy ses were conducted compared to the control.
The NOEC values, based on area under the growth curve, growth rate, and yield, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett's test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett's test, a Shapiro-Wilk's test and a Levene's test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk's and Levene's tests indicated normality and insignificant heterogeneity (i.e., p >0.01), the analysis was performed on the non- transformed raw data. In instances of non-normality or heterogeneity (i.e., p <0.01), a square root transformation was performed.

Table 1: Analytical Results: Measured concentrations of the test item in test solutions during the 96 h growth inhibition test with the Marine Diatom Skeletonema costatum

Nominal Concentration	Measured Concentration as mg a.i./L (Percent Nominal)
(mg a.i./L)	0 h	96 h	Geometric mean
0 (Control)	<MQLa	<MQLa	<MQLa
0.0051	0.00720 (141)b	0.00551 (108)	0.00630(124)
0.0051 abiotic	0.00720 (141)c	0.00682(134) d	0.00701 (137)
0.013	0.0131 (101)	0.00967 (74)	0.0113(87)
0.032	0.0324(101)	0.0247 (77)	0.0283 (88)
0.080	0.0736 (92)	0.0583 (73)	0.0655 (82)
0.20	0.189 (95) d	0.136 (68) d	0.160 (80)
0.50	0.467 (93)d	0.402 (80)	0.433 (87)

a MQL = 0.00370 mg a.i/L

b Re-diluted in duplicate and re-analyzed, mean of original and duplicate re-analyses reported.

c Reported value is result from level 1 hour 0.

d Re-diluted in duplicate and re-analyzed, mean of duplicate re-analyses reported.

e Average of three low spikes

Table 2: Cell density values for Skeletonema costatum during a 96 h exposure to the test item

Geometric Mean Concentration (mg a.i./L)	Mean Cell Density (cells/mL x 10E+04)				% Inhibition
	24 h	48h	72 h (%CV)	96 h (%CV)	72 h	96h
Control	3.64	8.25	22.0 (CV: 4)	63.0 (CV: 2)	—	—
0.00630	3.45	8.31	21.5 (CV: 5)	63.1 (CV: 2)	3	0
0.0113	3.39	8.00	19.9 * (CV: 2)	45.5 *(CV: 1)	10	28
0.0283	3.33	5.47 *	15.3 * (CV: 5)	39.5 *(CV: 4)	31	37
0.0655	3.28	5.09 *	12.3 * (CV: 5)	28.7 *(CV: 7)	44	54
0.160	2.22 *	2.84 *	4.86 * (CV: 4)	7.72* (CV: 11)	78	88
0.433	1.36*	1.61 *	2.70 * (CV: 6)	1.75 * (CV: 18)	88	97

* Significant reduction in cell density as compared to the control (Dunnett's test, p = 0.05)

Table 3: Growth rate values from time zero for Skeletonema costatum during a 96 h exposure to the test item

Geometric Mean Concentration (mg a.i./L)	Mean Growth Rate (cells/mL/hour)			%Inhibition
	0-24 h	0-48 h	0-72 h (% CV)	0-96 h (% CV)	72 h	96 h
Control	0.0536	0.0439	0.0429 (CV: 1)	0.0432 (CV: 0)	—	—
0.00630	0.0514	0.0441	0.0426 (CV: 2)	0.0432 (CV: 1)	1	0
0.0113	0.0507	0.0433	0.0415 * (CV: I)	0.0398 * (CV: 0)	3	8
0.0283	0.0500	0.0354 *	0.0379* (CV: 2)	0.0383* (CV: 1)	12	11
0.0655	0.0493	0.0338*	0.0348* (CV: 2)	0.0350* (CV: 2)	19	19
0.160	0.0327 *	0.0215 *	0.0220* (CV: 2)	0.0213* (CV: 5)	49	51
0.433	0.0125 *	0.00959 *	0.0138* (CV: 6)	0.00569* (CV: 35)	68	87

* Significant reduction in growth rate as compared to the control (Dunnett’s test, p = 0.05).

Table 4: Validity criteria

Table 4: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	63	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	19.4	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.44	Yes

Validity criteria fulfilled:

yes

Description of key information

Freshwater:

IC10 (72 h) 0.0605 mg a.i./L (growth rate, Navicula pelliculosa, geometric mean measured, OCSPP 850.4500, Bergfield 2015)

IC50 (72 h) 0.435 mg a.i./L (growth rate, Navicula pelliculosa, geometric mean measured, OCSPP 850.4500, Bergfield 2015)

IC10 (96 h) 0.0586 mg a.i./L (growth rate, Navicula pelliculosa, geometric mean measured, OCSPP 850.4500, Bergfield 2015)

IC50 (96 h) >0.471 mg a.i./L (growth rate, Navicula pelliculosa, geometric mean measured, OCSPP 850.4500, Bergfield 2015)

Marine water

IC10 (96 h) 0.0451 mg a.i./L (growth rate, Skeletonema costatum, geometric mean measured, OECD 201, Mays 2015b)

IC50 (96 h) 0.155 mg a.i./L (growth rate, Skeletonema costatum, geometric mean measured, OECD 201, Mays 2015b)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.435 mg/L

EC50 for marine water algae:

0.155 mg/L

EC10 or NOEC for freshwater algae:

0.06 mg/L

EC10 or NOEC for marine water algae:

0.045 mg/L

Additional information

Three experimental studies are available on the short-term toxicity to aquatic algae and cyanobacteria of the test item Chlorophene (CAS 120-32-1) (Bergfield 2015, Mays 2015a and Mays 2015b). Two studies were conducted with freshwater species Anabaena flos-aquae and Navicula pelliculosa, respectively and one with the marine species Skeletonema costatum. The studies were performed according to OCSPP guideline 850.4500 (in case of Navicula pelliculosa and Skeletonema costatum) and 850.4550 (in case of Anabaena flos-aquae). The nominal concentrations were verified by analytical monitoring. All studies were performed according to GLP.

For the key study (Bergfield 2015) the freshwater species Navicula pelliculosa was exposed under static conditions for 96 h to the following nominal concentrations (and the concentrations based on the geometric mean measured concentrations) Control, 0.0043 (0.00457), 0.011 (0.00990), 0.027 (0.0218), 0.067 (0.0569), 0.17 (0.153) and 0.50 (0.471) mg a.i./L. Since the measured concentration was within a range of 94% to 105% of nominal at test start and between 69 and 119% at test end (after 96h). Since the measured concentration was without the range of 80 to 120% of nominal the toxicological endpoints were evaluated using the nominal and actual concentrations (based on the geometric mean of each test item concentration). The IC50 was determined to be at 0.435 mg a.i/L, the IC10 value was at 0.0605 mg a.i./L and the NOEC at 0.0218 mg a.i./L after 72-h for growth rate. The IC50 was determined to be at >0.471 mg a.i/L, the IC10 value was at 0.0586 mg a.i./L and the NOEC at 0.0218 mg a.i./L after 96-h for growth rate.

In the study with Anabaena flos-aquae (Mays 2015a) the algae species was exposed under static conditions for approximately 96 h to the following nominal concentrations (and actual concentrations based on the geometric mean measured concentrations): Control, 0.031 (0.0263), 0.077 (0.0608), 0.19 (0.159), 0.48 (0.376), 1.2 (1.02) and 3.0 (2.77) mg a.i./L. Mean measured calculations were based on the recoveries of the test solutions on Days 0 and 4. Since the measured concentrations were in a range of 85% and 97% of nominal at test start and between 71 and 99% at test end (after 96 h) all toxicological endpoints were evaluated using the actual concentrations based on the geometric mean measured concentrations. After 72 h the IC50 for growth rate was determined to be at 0.989 mg a.i./L, an IC10 of 0.794 mg a.i./L and a NOEC of 0.159 mg a.i./L. After 96 h the IC50 for growth rate was determined to be at 1.11 mg a.i./L and an IC10 of 0.885 mg a.i./L. The NOEC was determined to be at 0.159 mg a.i./L after both time points.

The key study for marine water was performed with Skeletonema costatum (Mays 2015b). The species was exposed under static conditions for 96 hours to the following nominal concentrations (and actual concentrations based on the geometric mean measured concentrations): Control, 0.0051 (0.00630), 0.013 (0.0113), 0.032 (0.0283), 0.080 (0.0655), 0.20 (0.160) and 0.50 (0.433) mg a.i./L. Mean measured calculations were based on the recoveries of the test solutions on Days 0 and 4. Since the measured concentrations were in a range of 92% and 141% of nominal at test start and between 68 and 108% of nominal at test end (after 96 h) all toxicological endpoints were evaluated using the actual concentrations based on the geometric mean measured concentrations. After 96 h the IC50 for growth rate was determined to be at 0.155 mg a.i./L, an IC10 of 0.0451 mg a.i./L and a NOEC of 0.00630 mg a.i./L.

An additional study was performed with Pseudokichneriella subcapitata (SAG 61.81) with PREVENTOL BP technical (Egeler, P. et al. 2006, AN1AO). The test item concentration range from 0 (control), 0.0048, 0.0153, 0.0488, 0.1563 and 0.5 mg test item/L. Values are based on the overall GC/MS mean measured concentrations. A clear dose – response relationship can be derived from the cell concentration data. NOErC = 0.1036 mg/L; NOEbC = 0.0323 mg/L; ErC50 = 0.1972 mg/L; EbC50 = 0.1314 mg/L.