Clorofene

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 - 13 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.23, 0.35, 0.53, 0.80 and 1.2 mg a.i./L
- Sampling method: Samples were taken at test start and test end (48 h).
- Sample storage conditions before analysis: All samples were stored refrigerated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 0.010 mg a.i./mL primary stock solution was prepared at test initiation by diluting 0.0101 grams of the test item to a volume of 1,000 mL with freshwater. The test solutions were performed by appropriate dilutions of the stock solution
- Controls: water only

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: in-house daphnid culture
- Age of parental stock (mean and range, SD): 12 days
- Feeding during test: no feed during the test
- Food type: fed a suspension of the algal species Pseudokirchneriella subcapitata supplemented by an artificial diet consisting of a wheat grass, salmon starter, and yeast suspension.
- Frequency: at least once a day

ACCLIMATION
- Acclimation period: Since the culturing and testing environmental parameters were equivalent (i.e. temperature, dilution water, and lighting), no acclimation period was necessary .
- Acclimation conditions: same
- Health during acclimation: The adults were considered acceptable with no signs of stress, disease or physical damage.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Daphnia magna neonates (<24 h old) were used for the test. Neonates for the definitive test were collected from a single culture containing adults that were approximately 12 days old.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

146 mg CaC03/L

Test temperature:

19.9 - 20.4 °C (Control)
19.8 - 20.6 °C (Test item concetrations)

pH:

8.4 - 8.6 (Control)
8.4 - 8.6 (Test item concentrations)

Dissolved oxygen:

8.3 to 9.0 mg a.i/L (95 to 103% saturation)

Conductivity:

338 µS/cm

Nominal and measured concentrations:

0 (control,. 0.23, 0.35, 0.53, 0.80, and 1.2 mg a.i./L (nominal)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with a plastic dish)
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL volume.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). Prior to use. the dilution wrater was passed through a sediment 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)
- Total organic carbon: <2.0 mg/L
- Total hardness: 130 to 160 mg CaCO3/L
- Alkalinity: 156 mg CaCO3/L
- Ca/mg ratio: 61.6 / 28 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Total hardness, total alkalinity, and conductivity were measured in the dilution water at test initiation.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark
- Light intensity: 626 to 670 lux

EFFECT PARAMETERS MEASURED: The daphnids were observed for immobility and sublethal effects at approximately 24 and 48 h after test initiation.

RANGE-FINDING STUDY
- Test concentrations: Control, 0.010. 0.10, 0.50, 1.0 and 10 mg a.i./L.
- Results used to determine the conditions for the definitive study: 0, 0, 0, 0, 100, and 100% in the Control and 0.010, 0.10, 0.50, 1.0 and 10 mg a.i./L; based on these results the test item concentrations for the main test were choosen: Control, 0.23, 0.35, 0.53, 0.80 and 1.2 mg a.i./L

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.655 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limit

Remarks:

0.599 and 0.717 mg a.i./L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.443 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No
- Observations on body length and weight: No sublethal effects were noted during the definitive test.
- Mortality of control: 0
- Other adverse effects control: No
- Effect concentrations exceeding solubility of substance in test medium: The control and all treatment solutions were clear and colorless wiith no visible particulates, surface film, undissolved test substance, or precipitate throughout the test.

Table 1: Measured Concentrations as mg a.i./L (percent nominal) of test item during the 48-hour acute toxicity test with Daphnia magna

Nominal Concentration (mg a.i./L)	0-Hour	48-Hour	Mean Measured Concentration
Control	<MQLa	<MQLa	<MQLa
0.23	0.182 (79)	0.177 (77)	0.180(78)
0.35	0.280 (80)	0.277 (79)	0.279 (80)
0.53	0.437(82)	0.449 (85)	0.443 (84)
0.80	0.664 (83)	0.683 (85)	0.674 (84)
1.2	0.986 (82)	0.999 (83)	0.993 (83)
	QC Fortification Spikes (% Recovery)
Low Spike (0.0384)	0.0403 (105)	0.0408(106)	—
High Spike (2.40)	2.18(91)	2.26 (94)	—

MQLa = 0.00372 mg a.i./L

 

Table 2: Immobility of Daphnia magna exposed to the test item for 48-hours under static test conditions

Mean Measured Concentration (mg a.i./L)	Rep	Cumulative Percent Immobile (% Sublethal Effects)
		24 h	48 h	Treatment Mean Percent immobile (48 h)
0 (Control)	A	0(0)	0(0)	0
	B	0(0)	0(0)
	C	0(0)	0(0)
	D	0(0)	0(0)
0.180	A	0(0)	0(0)	0
	B	0(0)	0(0)
	C	0(0)	0(0)
	D	0(0)	0(0)
0.279	A	0(0)	0(0)	0
	B	0(0)	0(0)
	C	0(0)	0(0)
	D	0(0)	0(0)
0.443	A	0(0)	0(0)	0
	B	0(0)	0(0)
	C	0(0)	0(0)
	D	0(0)	0(0)
0.674	A	0(0)	60(0)	55*
	B	0(0)	80(0)
	C	0(0)	40(0)
	D	0(0)	40(0)
0.993	A	80(0)	100(0)	100*
	B	80(0)	100(0)
	C	80(0)	100(0)
	D	80(0)	100(0)

Note: Five daphnids were placed in each replicate at test initiation, totaling 20 daphnids per treatment.        

* Statistically significant compared to the control (Dunnett's one-tailed test; p - 0.05) 

Table 3: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
In the control, including the control containing the solubilising agent, not more that 10% of the daphnids should have been immobilized.	No immobility	Yes
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels.	8.3 to 9.0 me/L	Yes

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

05 - 09 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: controls and all test item concentrations
- Sampling method: sampled at test start and test end
- Sample storage conditions before analysis: All samples were stored refrigerated.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 0.010 mg a.i./mL primary standard solution was prepared at test initiation by diluting 0.0101 g of test item to a volume of 1,000 mL with dilution water. The primary stock solution was prepared using sonication with heat for approx two hours prior to prepare the series of test concentrations. The test solutions were prepared by diluting appropriate volumes of the primary standard solution to a volume of 1.0 L with dilution water for final concentrations of 0.033, 0.065, 0.13, 0.25, and 0.50 mg a.i./L.
- Controls: dilution water only.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid Shrimp
- Age of parental stock (mean and range, SD): Adults (5 to 6 days old)
- Feeding during test: yes
- Food type: live brine shrimp (Artemia sp) nauplii
- Frequency: at least once per day
- Age at study initiation: ≤ 24 h

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Post-larvae (<24 hours old) mysid shrimp were used for the test contained mysids that were (5 to 6 days old) at test initiation. The individuals within each container were transferred via glass pipet below solution surface from each container into the corresponding test chamber.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

130 to 160 mg CaCO3/L

Test temperature:

24.4 - 24.6 °C (Control)
24.4 - 24.9 °C (Test item concentrations)

pH:

8.1 - 8.2 (Control)
8.0 - 8.3 (Test item concentrations)

Dissolved oxygen:

6.1 - 7.1 mg/L (Control)
6.1 - 7.2 mg/L (Test item concentrations)
Note: 100% oxygen saturation values in saltwater with salinity of 20‰ are 7.3 and 7.2, at 24 and 25 °C, respectively

Salinity:

19.8 - 20.2 ‰ (Control)
19.8 - 20.2 ‰ (Test item concentrations)

Nominal and measured concentrations:

Control, 0.033, 0.065, 0.13, 0.25 and 0.50 mg a.i./L (nominal)
Control, 0.0252, 0.0503, 0.108, 0.208 and 0.402 mg a.i./L (mean measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open (covered with a plastic Petri dish)
- Material, size, headspace, fill volume: 500-mL glass jars containing approx 250 mL of control or test solution.
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix to laboratory freshwater at a target salinity of 20 ± 3‰. The laboratory freshwater consists of well water blended with well water that was demineralized by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO3/L. Prior to use, the dilution water was treated with UV irradiation and passed through a 1 µm filter.
- 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; Magnesium: 643 mg/L; Potassium: 210 mg/L; Sodium: 5720 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: Temperature, dissolved oxygen, pH, and salinity were measured at initiation, at observance of 100% mortality, and at termination in the replicate test chambers of all treatments during the definitive test.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark with a 30 min simulated dawn and dusk periods
- Light intensity: 626 lux

EFFECT PARAMETERS MEASURED: Observations for mortality and sublethal responses were made once every 24 hours (±1 hour) for the duration of the test. The criterion for the effect was death (animals exhibiting no response to a physical stimulus).

RANGE-FINDING STUDY
- Test concentrations: Control, 0.0010, 0.010, 0.050, 0.10, and 0.50 mg a.i./L
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test it was determined that post-larvae mysid shrimp would be used for the definitive test.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.089 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% confidential limits

Remarks:

0.0740 - 0.106 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Behavioural abnormalities: lethargy observed at 0.108 and 0.402 mg/L after 24 h
- Mortality of control: no

Validity criteria concerning OPPTS 850.1035

Control animals (100% survival during the definitive test) satisfied test acceptability criteria for survival (i.e., > 90%) as stated in the study protocol and the OPPTS 850.1035 testing guideline.

Table 1: Measured concentrations of the test item during the 96-h acute toxicity test with Americamysis bahia

Nominal Concentrations (mg a.i./L)	Measured Concentration as mg a.i./L (percent nominal)
	0 h	96h	Arithemtic mean
Control	<MQL	<MQL	<MQL
0.033	0.0293 (89)	0.0210 (64)	0.0252 (76)
0.065	0.0580 (89)	0.0426 (66)	0.0503 (77)
0.13	0.119 (92)	0.0973 (75)	0.108 (83)
0.25	0.220 (88)	0.196 (78)	0.208 (83)
0.50	0.400 (80)	0.404 (81)	0.402 (80)
Stock (Percent Recovery)
10	9.78 (98)	---	---
QC Fortifications (Percent Recovery)
Low Spike (0.0192)	0.0196 (102)	0.0211 (110)	---
High Spike (0.720)	0.675 (94)	0.752 (104)	---

 

Table 2: Mortality of Mysid Shrimp, Americamysis bahia, exposed to test item for 96 h under static conditions

Mean Measured Concentration (mg a.i./L)	Rep	Cumulative Number Dead (%Mortality)
		24 h	48 h	72 h	96 h	Treatment Mean
Control	A	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
	B	0 (0)	0 (0)	0 (0)	0 (0)
	C	0 (0)	0 (0)	0 (0)	0 (0)
	D	0 (0)	0 (0)	0 (0)	0 (0)
0.0252	A	0 (0)	0 (0)	0 (0)	0 (0)	0(0)
	B	0 (0)	0 (0)	0 (0)	0 (0)
	C	0 (0)	0 (0)	0 (0)	0 (0)
	D	0 (0)	0 (0)	0 (0)	0 (0)
0.0503	A	0 (0)	0 (0)	0 (0)	0 (0)	1(5)
	B	0 (0)	0 (0)	0 (0)	0 (0)
	C	0 (0)	0 (0)	0 (0)	0 (0)
	D	0 (0)	0 (0)	1 (20)	1 (20)
0.108	A	0 (0)a	3 (60)	4 (80)	4 (80)	14 (70)*
	B	0 (0)	4 (80)	4 (80)	4 (80)
	C	0 (0)	2 (40)	3 (60)d	3 (60)
	D	0 (0)	2 (40)	3 (60)d	3 (60)
0.208	A	2 (40)	4 (80)	5 (100)	5 (100)	20 (100)*
	B	2 (40)	4 (80)	5 (100)	5 (100)
	C	1 (20)	4 (80)	5 (100)	5 (100)
	D	0 (0)	4 (80)	5 (100)	5 (100)
0.402	A	3 (60)a	5 (100)	5 (100)	5 (100)	20 (100)*
	B	4 (80)	5 (100)	5 (100)	5 (100)
	C	4 (80)b	5 (100)	5 (100)	5 (100)
	D	4 (80)b	5 (100)	5 (100)	5 (100)

* Statistically significant different to the control

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 14 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition test))

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control, 0.024, 0.042, 0.076, 0.14, 0.25, 0.44 and 0.80 mg a.i./L
- Sampling method: sampled at test start (0 h), after 48 h and at test end (96 h)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
A stock solution was prepared at a target concentration of 100 mg a.i./L by diluting 0.9100 g of the test item to 9.0 L with dilution water. Additional diluter stock solutions were prepared at a target concentration of 100 mg a.i./L by diluting 0.04044 or 0.9100 g of the test item to 4.0 L or 9.0 L with dilution water. Following the addition of the dilution water, the test substance was crushed with a glass stir rod; heated sonicator for approximately two hours and followed by a heated stirring procedure until the remainder of the test substance was in solution. Stock solutions were kept stirring at room temperature for the duration of their use. For each cycle of the diluter system, two pumps were used to deliver appropriate volumes of stock solution from a single reservoir to two separate mixing boxes. The 0.024, 0.042, 0.076, 0.14, 0.25, and 0.44 mg a.i./L test substance treatments were prepared by injecting 19.8 mL of the 100 mg a.i./L stock solution into a 4.5 L volume of dilution water, resulting in a 0.44 mg a.i./L solution in the main chemical mixing box. The 0.44 mg a.i./L solution was used directly for the 0.44 mg a.i./L test solution, while the lower treatment levels were prepared via serial dilution using the diluter system. The 0.80 mg a.i./L test solution was prepared by injecting 16.0 mL of the 100 mg a.i./L stock solution into a 2.0 L volume of dilution water in a separate mixing box, resulting in the 0.80 mg a.i./L test solution.

- Controls: test medium only

Test organisms (species):

other aquatic mollusc: Crassostrea virginica

Details on test organisms:

TEST ORGANISM
- Common name: Eastern oyster
- Justification for species other than prescribed by test guideline: selected because of the availability of the species
- Source: obtained from Circle C Oyster Ranch, Ridge, Maryland, USA
- Feeding during test: yes
- Food type: marine microalgal concentrate (Instant Algae Shellfish Diet 1800, Reed Mariculture, Inc.)
- Amount: each test chamber: at least 10 mL was added during exposure with exceptions of test initiation when 10 mL was added twice, and termination, when 10 mL was added only once.
- Frequency: three times each day

ACCLIMATION
- Acclimation period: 3 days
- Acclimation conditions: laboratory saltwater at approximately 20‰ salinity and 20 °C, water was continuously re-circulated and aerated
- Other: Prior to placement of oysters in aquaria for acclimation, approximately three to five mm of the shell periphery at the ventral end was ground away with an electric disc grinder.
- Type and amount of food: Instant Algae Shellfish Diet 1800 (supplied by Reed MariCulture, Inc.), a marine microalgae concentrate
- Feeding frequency: at least once each day
- Health during acclimation (any mortality observed): No mortalities were observed during the acclimation period


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Oysters used for the definitive test ranged from 32.3 to 47.2 mm in valve height (mean and standard deviation = 38.3 ±4.1 mm) as measured on a representative group of oysters from the test population on the day of test initiation. A representative group (n = 5) of animals were sacrificed prior to study initiation for the assessment of the gonadal development and pre-spawn condition. Visual, i.e., unaided, and microscopic observations were performed on these animals and it was determined that none of the animals were in spawning condition.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20.6 - 20.9 °C (Control)
20.5 - 20.9 °C (Test item concetrations)

pH:

8.0 - 8.2 (Control and test item concentrations)

Dissolved oxygen:

6.3 - 7.4 mg/L (81 - 95% saturation) (Control)
6.1 to 7.6 mg/L (78 to 97% saturation) (Test item concentrations)

Salinity:

19.0 - 19.1 ‰ (Control)
18.9 - 19.2 ‰ (Test item concentrations)

Nominal and measured concentrations:

Control, 0.024, 0.042, 0.076, 0.14, 0.25, 0.44 and 0.80 mg a.i./L (nominal)
Control, 0.0135, 0.0224, 0.0423, 0.0803, 0.142, 0.254 and 0.621 mg a.i./L (mean measured concentrations)

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: Glass aquaria measuring approx 21.7 cm in width, 37.0 cm in length, and 17.8 cm in height; the depth of the test solution was maintained at approx 10.5 cm and the volume of each test chamber was approx 8.4 L.
- Type of flow-through:
The diluter system used for the definitive test delivered approximately 2,000 mL of solution with each cycle, with this volume being equally divided between replicate test chambers for a given treatment (i.e., approximately 1,000 mL was delivered to the appropriate replicate test chamber at each cycle). During the in-life phase of the definitive test, an average of 118 L per day of control dilution water and test substance treatment solution was delivered to each chamber. The accuracy of the diluter was verified by volumetric measurement before test initiation and the diluter was allowed to operate approximately three days prior to initiating the definitive test. Proper operation of the proportional diluter and all mechanical systems was verified twice each day during the definitive test. Each test chamber contained a small pump to continuously recirculate test solution; Solution delivery from the diluter and flow generated by the re-circulating pumps within each test chamber were sufficient to meet the test guideline flow rate requirements as demonstrated by the successful growth of the control animals during the test. The test chambers were immersed in a circulating water bath that was maintained at approximately 20°C.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory saltwater used as culture and dilution water in this study was prepared at a target salinity 20 ± 2 ‰ by adding a commercial sea salt mix (Crystal Sea Marinemix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater blended with well water that was demineralized by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO3/L. Prior to use, the dilution water was heated, passed through a 1 µm filter and an ultraviolet sterilizer.
- 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 solution temperature, pH, dissolved oxygen concentration, and salinity were measured daily in each test chamber.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark photoperiod with a 30 min simulated dawn and dusk transition period
- Light intensity: 464 to 657 lux

EFFECT PARAMETERS MEASURED: Observations for mortality and other signs of test substance effect were made daily. New shell growth at test termination was measured to the nearest 0.1 mm with a vernier caliper.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.286 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Remarks on result:

other: 95% confidential limits

Remarks:

0.250 - 0.322 mg a.i./L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Details on results:

- Other biological observations: There was a noticeable reduction in fecal material observed in the 0.254 and 0.621 mg a.i./L test substance treatments as compared to the control throughout the test. Mean new shell growth was 2.8, 2.6, 2.4, 2.2, 2.1, 2.0, 1.7, and 0.4 mm in the control, 0.0135, 0.0224, 0.0423, 0.0803, 0.142, 0.254, and 0.621 mg a.i./L treatments, respectively; percent change in new shell growth ranged from 8% in the 0.0135 mg a.i./L treatment to 87% in the 0.621 mg a.i./L treatment, as compared to the control mean new shell growth (i.e., 2.8 mm).
- Mortality of control: no

Reported statistics and error estimates:

All statistical analyses were performed with SAS software (version 9.3). A one-tailed Dunnett’s test was conducted at the 0.05 level of significance, with comparisons made to the control group. The alternate hypothesis was that the mean new shell growth for the test substance treatment group had been reduced in comparison to the control mean new shell growth. Prior to the Dunnett’s test, a Bartlett’s test for homogeneity of variance over treatments was conducted. The assumptions of normality and homogeneity of variance were met for the raw data values; therefore, a parametric analysis was performed on the raw data. The EC50 for new shell growth was estimated by a four-parameter logistic (sigmoid-shaped) model, two parameters fixed (100 and 0% inhibition), fit to the data with percent inhibition as the dependent variable and log concentration as the independent variable.

Table 1: Mean new shell growth exhibited by Eastern Oysters (Crassostrea virginica) after 96 h of exposure to the test item

Mean Measured Concentration	Shell Growth Statistics
(mg a.i./L)	Mean Length ±SD (mm)	Percent Difference From Control
0 (control)	2.8 ±0.7 (range: 1.8 to 4.1)	NA
0.0135	2.6 ±0.4 (range: 2.0 to 3.6)	8.2
0.0224	2.4 ±0.6 * (range: 1.4 to 3.6)	16
0.0423	2.2 ±0.6 * (range: 1.2 to 3.4)	24
0.0803	2.1 ±0.6 * (range: 1.3 to 4.1)	24
0.142	2.0 ±0.4 * (range: 1.3 to 3.1)	28
0.254	1.7 ± 0.6 * (range: 0.8 to 3.3)	38
0.621	0.4 ±0.5 * (range: 0.0 to 1.2)	87

  * Statistical significant difference between the control (Dunnett`s Test, p < 0.05). The 96 h EC50 was estimated to be 0.286 mg a.i./L (95% confidence limits of 0.250 and 0.322 mg a.i./L). The 96 h NOEC was 0.0135 mg a.i./L.

Table 2: Measured concentrations of the test item during a 96 h exposure of the eastern Oyster, Crassostrea virginica, under flow-through conditions

Nominal Concentration (mg a.i./L)	Measured Concentration as mg a.i./L (Percent Nominal)
	Day -2	0 h	48 h	96 h	Mean Measured Concentration a	Geometric Mean Measured Concentration a
Control	<MQL b	<MQL b	--	<MQL b	<MQL b	<MQL b
0.024	0.00770 (32)	0.0154 (64)	0.0129 (54)	0.0123 (51)	0.0135 (56)	0.0135 (56)
0.042	0.0127 (30)	0.0229 (55)	--	0.0219 (52)	0.0224 (53)	0.0224 (53)
0.076	0.0218 (29)	0.0445 (59)	0.0388 (51)	0.0435 (57)	0.0423 (56)	0.0422 (56)
0.14	0.0614 (44)	0.0783 (56)	--	0.0823 (59)	0.0803 (57)	0.0803 (57)
0.25	0.0743 (30)	0.138 (55)	0.137 (55)	0.151 (60)	0.142 (57)	0.142 (57)
0.44	0.137 (31)	0.244 (55)	--	0.263 (60)	0.254 (58)	0.253 (58)
0.80	0.590 (74)	0.635 (79)	0.628 (79)	0.599 (75)	0.621 (78)	0.620 (78)
100 (Stock Solution)	94.0 (94)	98.3 (98)	--	93.1 (93)	95.7 (96)	95.7 (96)
QC Fortification Spikes (% Recovery)
Low Spike (0.0192)	0.0181 (94)	0.0200 (104)	0.0172 (90)	0.0107 (111) c	--	--
High Spike (1.20)	1.10 (92)	1.18 (98)	1.17 (98)	1.08 (90)	--	--

a) Day -2 not included in calculations. b) MQL = 0.00372 mg a.i./L

c) Sample was re-diluted in triplicate. The average of the re-analysis is reported.

Description of key information

Freshwater:

EC50 (48h) = 0.655 mg a.i./L (Lamichhane 2015a, Daphnia magna, measured, OECD 202)

Marine Water:

EC50 (96h) = 0.089 mg a.i./L (Lamichhane 2015b, Americamysis bahia, measured, EPA OPPTS 850.1035)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.655 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.089 mg/L

Additional information

Three experimental studies are available on the short-term toxicity to aquatic invertebrates of the test item Chlorophene (CAS120-32-1) (Lamichhane 2015a, Lamichhane 2015b, VanHooser 2015). One study was conducted with the freshwater species Daphnia magna and two with the marine species Americamysis bahia and Crassostrea virginica, respectively. The studies were performed according to OECD guideline 202 (in case of Daphnia magna), according to EPA OPP 72-3 (in case of Americamysis bahia) and additionally according to OPPTS 850.1035 (in case of Crassostrea virginica). The nominal concentrations were verified by analytical monitoring. All studies were performed according to GLP.

The key study for freshwater (Lamichhane 2015a) was conducted with Daphnia magna. The test organism was exposed over a period of 48 h to nominal concentrations of 0, 0.23, 0.35, 0.53, 0.80 and 1.2 mg a.i./L (nominal) and to actual concentrations of 0.180, 0.279, 0.443, 0.674 and 0.993 mg a.i./L (based on mean measured concentrations). Juvenile daphnids (less than 24 h old) were exposed without feeding in a static system. All treatments were in triplicate with 10 daphnids per test vessel. Samples of the freshly prepared test solutions were taken for analysis of the test item at test start 0 h and at test end after 48 h were analyzed. The mean measured test item concentration at test start was between 79 to 83% of nominal and between 77 to 85% of nominal at test end. Therefore, all toxicity values were based on the nominal concentrations and based on mean measured concentrations. The EC50 was determined to be at 0.655 mg a.i./L.

The key study for the marine compartment (Lamichhane 2015b) was conducted with the mysid shrimp Americamysis bahia. The test species was exposed over a period of 96 h to the nominal (mean measured) concentrations of 0 (control), 0.033 (0.0252), 0.065 (0.0503), 0.13 (0.108), 0.25 (0.208) and 0.50 (0.402) mg a.i./L in natural filtered seawater (pH at 8.0-8.3, salinity up to 20.2 ‰) for a 96 h period. All treatments were assayed in duplicates with 10 mysids per test vessel. There was 100% survival among control organisms during the course of the study and all control organisms appeared normal. Therefore, the control group satisfied test acceptability criteria for survival (i.e., > 90%) as stated in the study protocol and the OPPTS 850.1035 testing guideline. The 96-h LC50 value was estimated to be 0.0890 mg a.i./L (95% confidence limits: 0.0740 and 0.106 mg a.i./L) based on mean measured concentrations. The only sublethal effect observed was lethargy in the 0.108 and 0.402 mg a.i./L test substance concentrations at 24 h. The slope of the 96 -h concentration response line was 6.9 (95% confidence limits: 4.0 and 9.8). The 96-h NOEC was 0.0503 mg a.i./L based on mean measured concentrations and a lack of significant mortality and sublethal effects at this and all lower test substance treatments. The measured concentrations were within a range of 80 to 120% of nominal for test start and between 64 to 81% of nominal at test end. Based on these results the toxicological endpoints were evaluated using the nominal and mean measured concentration.

The second study for the marine compartment (VanHooser, 2015) was conducted with eastern oysters (Crassostrea virginica) under flow-through conditions.The test species was exposed to nominal concentrations (and mean measured concentrations) of control (0), 0.024 (0.0135), 0.042 (0.0224), 0.076 (0.0423), 0.14 (0.0803), 0.25 (0.142), 0.44 (0.254) and 0.80 (0.621) mg a.i./L for 96 h. The dilution water was natural filtered sea water with a salinity of up to 19.2 ‰ and a pH of up to 8.2. Reduction of shell deposition was used as the indicator of toxicity. The measured concentration ranged between 55 to 79% of nominal at test start and between 51 to 75% at test end (after 96 h). Based on these results the toxicological endpoints were evaluated using the nominal and actual (mean measured) concentrations. The mean survival (100%) and the mean value of new shell growth (2.8 mm) of the control treatment were greater than the survival (90%) and growth (> 2 mm) acceptability criteria set by the study protocol and the OPPTS 850.1025 testing guideline. Based on mean measured concentrations of Ortho-benzyl-para-chlorophenol, the 96-hour EC50 was 0.286 mg a.i./L (95% confidence limits of 0.250 and 0.322 mg a.i./L) and the 96-h NOEC was 0.0135 mg a.i./L, based on the lack of a statistically significant reduction in new shell growth at this and all lower concentrations.