Octahydro-4,7-methano-1H-inden-5-yl acetate

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:

16 March 2010 and 15 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.

Justification for type of information:

The information of Cyclacet is used for read across to Cyclacet Dihydro (for further details see the Endpoint summary).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations
Water samples were taken from the control and each test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
The method of analysis, stability, recovery and test preparation analyses are described in Appendix 3 see attached section.

Vehicle:

no

Details on test solutions:

Range-finding test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test item was dissolved directly in water.
An amount of test item (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 25 minutes and the volume adjusted to 500 ml to give the 100 mg/l test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/l.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20°C to 22°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
A single temperature at the start of the test was observed to be in excess of the 20 ± 1°C stated in the Study Plan This was considered not to have affected the outcome or validity of the test as the temperature was within test guideline specification.
The control group was maintained under identical conditions but not exposed to the test item.

Experimental preparation
For the purpose of the definitive test the test item was dissolved directly in water.
An amount of test item (200 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 2 litres to give the 100 mg/l test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 56, 32, 18 and 10 mg/l.

Physico-chemical measurements
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 0039/1133) conducted approximately every six months used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
An amount of reference item (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 20°C.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Organism
-Common name:
Water flea

-Source:
Derived from in house laboratory cultures.

-Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.
These young were removed from the cultures and used for testing.

-Feeding no food during exposure

ACCLIMATION
-Acclimation period:
Not stated

-Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximatley 2 litres of reconstituted water in a temperature controlled room at approximately 20 degC.
The lighting cycle was controlled to give a 16 hour light and 8 hour darkness cycle with 20 minute dawn and dusk transition periods.

-Type and amount of food:
Each culture was fed daily with a suspension of algae (Chorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

-Health during acclimation:
No mortality observed

Test water
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at approximately 20°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.
See Appendix 4 for results in any other information on materials and methods section.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See Appendix 4 for results in any other information on materials and methods section.

Salinity:

freshwater used

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test item was dissolved directly in water.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Details on test conditions:

Exposure conditions
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 20°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL 21-29mg/l

Details on results:

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.

Definitive Test
Immobilisation data
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:
Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 28 24 - 33
48 25 21 - 29
The No Observed Effect Concentration after 24 and 48 hours exposure was 10 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 hours were 6.4 (SE = 1.2) and 6.8 (SE = 1.3) respectively.

Observations on test item solubility
All control and test concentrations were observed to remain clear colourless solutions throughout the duration of the test.

Verification of test concentrations
Analysis of the test preparations at 0 and 48 hours (see Appendix 3) showed measured test concentrations to range from 80% to 112% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

Positive Control
Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given in Table 3. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 3 and 4.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:
Time (h)
EC50 (mg/l) 95% Confidence limits
(mg/l)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72
The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.
The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
The EC50 value and associated confidence limits at 24 hours and the slope of the response curve and standard error were calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999). The EC50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	10	10

Table2              Cumulative Immobilisation Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	1	2	3	15	2	2	4	20
32	6	5	11	55	8	7	15	75
56	10	10	20	100	10	10	20	100
100	10	10	20	100	10	10	20	100

R₁– R₂= Replicates 1 and 2

Table3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	9	19	95	10	10	20	100
3.2	10	10	20	100	10	10	20	100

 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 25 mg/l with 95% confidence limits of 21 - 29 mg/l. The No Observed Effect Concentration at 48 hours was 10 mg/l.

Executive summary:

The acute toxicity of Cyclacet towards Daphnia magna was investigated according to OECD Guideline 202 under GLP. Daphnids were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg/l under static conditions and onserved for 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 80% to 112% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only. The 48h-EC50 was found to be 25 mg/l with 95% confidence limits of 21-29 mg/l.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02/08/2010 -06/08/2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study according to international guideline (OECD guideline 202) under GLP. Klimisch 2 is derived because the presence of oily slick in the test concentrations where immobility was also seen may have resulted in a more wors case EC50 value.

Justification for type of information:

This information is used to derive the acute / chronic ratio for Cyclaprop, which can then be applied to Cyclacet Dihydro (see for further details the Endpoint summary).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 11, 17, 25, 37 and 56 mg/L (nominal)
- Sampling method: Samples were collected from one test chamber of each treatment and control group one day prior to the start of the test after conditioning the diluter for approximately 18 hours. Samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test and at 48 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual stock solutions were prepared for each of the five concentrations tested. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 560 mg/mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 110, 170, 250 and 370 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and appeared clear and colorless. Stock solutions were placed in 60-mL syringes and placed on syringe pumps in the study room.
The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 5 µL/minute where they were mixed with dilution water delivered at a rate of 50 mL/minute to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all Cyclaprop treatment groups was 0.1 mL/L.
- Controls: Yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HPLC-grade dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution): 0.1 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): stock solutions appeared clear and colorless. Test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in the water column of any control or treatment solution. At test initiation, an oily-slick was noted on the surface of the solutions in the mixing chambers of the 25 (meas. 11), 37 (meas. 14.0) and 56 (meas. 7.2) mg/L test concentrations. At test termination, an oily-slick was noted on the surface of the solutions in the mixing chambers of all treatment groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: cultures maintained by Wildlife International, Ltd, Easton, Maryland.
- Age at study initiation (mean and range, SD): less than 24 hours old
- Method of breeding: Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2 week period immediately preceding the test, water temperatures in the cultures ranged from 18.8 to 20.5ºC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.1 to 8.5, measured with a Thermo Orion Benchtop pH/ISE Meter Model 4 Star Plus pH meter. Dissolved oxygen ranged from 7.7 to 9.1 mg/L (>=86% of saturation), measured with a Yellow Springs Instruments Model 85 dissolved oxygen meter.
Daphnids in the cultures were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. The adults were fed prior to test initiation, but neonates were not fed during the test.

The five adult daphnids used to supply neonates for the test were held for at least 25 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7 day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 10 daphnids. Each group of daphnids then was transferred to the test compartment in an indiscriminately assigned test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.
- Feeding during test: no feeding during the test


ACCLIMATION
- Acclimation conditions (same as test or not): same as test

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

138 mg/L as CaCO3

Test temperature:

20 +/- 1 degrees Celsius

pH:

8.2-8.3

Dissolved oxygen:

>= 7.6 mg/L (>= 84% of saturation)

Nominal and measured concentrations:

Nominal: 11, 17, 25, 37 and 56 mg/L
mean measured: 6.1, 7.2, 11, 14 and 7.2 mg/L

Measured concentrations of the samples ranged from approximately 10.6 to 59.9% of nominal (attached Table 1). Results of analyses to measure concentrations of Cyclaprop in the test solution samples collected during the test are presented (attached Table 2). Measured concentrations of the samples ranged from approximately 11.2 to 55.2% of nominal. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 6.1, 7.2, 11, 14 and 7.2 mg/L, representing 55, 42, 44, 38 and 13% of nominal concentrations, respectively. The relatively high volatility of the test substance resulted in analytical recoveries that were routinely below 60% in all treatment groups. The further decreases observed at the two highest concentrations were attributed to the limited solubility of the test substance in the test system. A flow-through test design was used to provide continuous delivery of the test substance to maximize concentrations. While overall recoveries were below 60%, concentrations were consistently maintained at each concentration, with coefficients of variation in analytical recoveries during the exposure ranging from 0.35 to 18%. Therefore, the results of the study were based on the mean measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance, a solvent control and a negative control (dilution water) to test chambers. Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. After mixing, the flow from each mixing chamber was split to deliver test water to two replicate test chambers.

The syringe pumps used to deliver stock solutions or solvent to the mixing chambers, and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the two replicates. Delivery of test solutions to the test chambers was initiated two days prior to test initiation in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.

The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period. Test chambers were 9 L glass aquaria filled with 7 L of test water. The depth of the test water in a representative chamber was 15.2 cm. Each test chamber contained one test compartment constructed from a glass beaker approximately 6.5 cm in diameter and 12 cm in height, with nylon screen attached to two holes on the sides of the beaker. The depth of the test water in a representative compartment was 7.0 cm. All test chambers were labeled with the project number, test concentration and replicate designation.

- Type (delete if not applicable): closed
- 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: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 m, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 m to remove fine particles and was passed through an ultraviolet (UV) sterilizer.

The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity and pH of the well water during the four-week period immediately preceding the test are presented. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water are presented.

- Total organic carbon: < 1 mg C/l
- Alkalinity: 182 mg/l as CaCO3
- Conductivity: 337 uS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured at the end and start of the experiment; pH and oxygen were measured at the beginning of the experiment and after 24 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: Measurements of pH ranged from 8.2 to 8.3.
- Photoperiod / Light intensity: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30 minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006C light meter. Light intensity at test initiation was 113 lux at the surface of the water of one representative test chamber.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility (after 2, 24 and 48 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Daphnia in the negative and solvent control groups generally appeared normal throughout the test, with the exception of one immobile daphnid noted in the solvent control at test termination. Two negative control daphnids were also noted floating at initial observations on the day of test initiation, but were normal in appearance after being gently submerged. Percent immobility at test termination in the 6.1, 7.2 (nominally 17 mg/L), 11, 14 and 7.2 mg/L (nominally 56 mg/L) treatment groups was 0, 5, 0, 20 and 20%, respectively. Floating daphnids were noted in all treatment groups, but were normal in appearance after being gently submerged. Floating daphnids were noted in the treatment groups throughout the test and at much higher frequencies than noted in the negative control, with all daphnids in some treatment groups noted as floating.

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

There was less than 50% immobility in any of the Cyclaprop treatment groups during the test, which precluded the statistical calculation of EC50 values at 24 and 48 hours. Therefore, the EC50 values were estimated to be greater than the highest concentration tested.

Measured concentrations of Cyclaprop in test solution samples

Nominal test concentration (mg/l)	Sample number (558A-105-)	Sampling time (days)	Measured concentration^1(mg/l)	Percentage of nominal^2	Mean measured concentration (mg/l)	Mean percent of nominal
Negative Control 0.0	1 8	0 2	<LOQ <LOQ	- -	-	-
Solvent Control 0.0	2 9	0 2	<LOQ <LOQ	- -	-	-
11	3 10	0 2	6.07 6.04	55.2 54.9	6.1	55
17	4 11	0 2	7.70 6.74	45.3 39.7	7.2	42
25	5 12	0 2	10.1 11.6	40.4 46.4	11	44
37	6 13	0 2	12.4 15.2	33.6 41.2	14	38
56	7 14	0 2	6.27 8.15	11.2 14.5	7.2	13

 

¹The limit of quantification (LOQ) was 2.02 mg/l, calculated as the product of the concentration of the lowest calibration standard (0.200 mg a.i./L) and the dilution factor of the matrix blank samples (10.0), corrected for the test substance purity (98.9%).

²Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly. Cumulative immobility and observations

Nominal concentration (mg/)	Rep.	Daphnia/replicate	~5 Hours		24 Hours		48 Hours		Percent immobile
			Number immobile^1	Observations^2	Number immobile^1	Observations^2	Number immobile^1	Observations^2
Negative control	A B	10 10	0 0	1 Q,AN; 9 AN 1 Q,AN; 9 AN	0 0	10 AN 10 AN	0 0	10 AN 10 AN	0
Solvent control	A B	10 10	0 0	10 AN 10 AN	0 0	10 AN 10 AN	0 1	10 AN 9 AN	5
11	A B	10 10	0 0	7 Q,AN; 3 AN 1 Q,AN; 9 AN	0 0	3 Q,AN; 7 AN 7 Q,AN; 3 AN	0 0	10 AN 4 Q,AN; 6 AN	0
17	A B	10 10	0 0	6 Q,AN; 4 AN 7 Q,AN; 3 AN	0 0	4 Q,AN; 6 AN 6 Q,AN; 4 AN	1 0	4 Q,AN; 5 AN 5 Q,AN; 5 AN	5
25	A B	10 10	0 0	8 Q,AN; 2 AN 10 AN	0 0	6 Q,AN; 4 AN 7 Q,AN; 3 AN	0 0	6 Q,AN; 4 AN 9 Q,AN; 1 AN	0
37	A B	10 10	0 0	10 AN 10 AN	0 0	8 Q,AN; 2 AN 9 Q,AN; 1 AN	2 2	6 Q,AN; 2 AN 4 Q,AN; 4 AN	20
56	A B	10 10	0 0	3 Q,AN; 7 AN 1 Q,AN; 9 AN	0 0	2 Q,AN; 7 AN 1 Q,AN; 9 AN	3 1	4 Q,AN; 3 AN 3 Q,AN; 6 AN	20

¹Cumulative number of immobile daphnids

²Observed effects: AN = appear normal; Q,AN = trapped at water surface but appear normal after gentle submersion

Validity criteria fulfilled:

yes

Remarks:

Oxygen saturation above 3 mg/l; mortality in controls < 10%

Conclusions:

The acute toxicity (48h-EC50) of Cyclaprop towards Daphnia magna is > 14 mg/l.

Executive summary:

The acute toxicity of Cyclaprop towards Daphnia magna was investigated according to OECD Guideline 202 under GLP. Daphnids were exposed to nominal concentrations of 11, 17, 25, 37 and 56 mg/l under flow-through conditions and observed for 48 hours. Based on measured concentrations, the 48h-EC50 was found to be >14 mg/l, the maximum solubility in the test medium obtained in this test. This is based on the fact that at test initiation, an oily-slick was noted on the surface of the solutions in the mixing chambers of the higher test concentrations. At test termination, this phenomenon as observed for all treatment groups. Presence of the oily-slick appears to indicate that test concentrations exceeded functional solubility in the test system. In addition, floating daphnids were noted in the treatment groups throughout the test and at much higher frequencies than noted in the negative control. This floating is indicative for the presence of oil. Despite the uncertainties on whether the immobility is physical or systemic the EC50 is considered to be a worst case concentration. In view of the efforts made further testing is not expected to change this conclusion.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The information is based on read across

Justification for type of information:

The full read across justification is presented in the Endpoint summary, the accompanying file is also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of Cyclacet Dihydro is directly derived from Cylacet using read across. This results in an EC50 for Cyclacet Dihydro of 25 mg/l.

Description of key information

The acute toxicity for Daphnia is 25 mg/l using read across from Cyclacet tested according to OECD TG 202.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

25 mg/L