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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04/06/2012 - 10/07/2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study according to international guideline (OECD guideline 211) under GLP. Reliability 2 is assigned because some oily slick was seen in the test chambers, indicating some insoluble test material and therefore physical effects potentially have influenced the results (see discussion)

Justification for type of information:

The information is used to derive the acute / chronic ratio for Cyclaprop which is then applied to Cyclacet Dihydro (see for justification the Endpoint summary).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Signatures are lacking (draft report)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.11, 0.33, 1.0, 3.0 amd 9.0 mg/l (nominal)
- Sampling method: Samples were collected from each treatment and control group approximately 27 hours prior to the start of the test after conditioning the diluter for approximately 18 hours. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at weekly intervals during the test and at the end of the test to measure concentrations of the test substance. All samples were collected from mid-depth, placed in 40 mL glass vials with septa. Samples were acidified with 10% phosphoric acid (10% H3PO4), and processed immediately for analysis.

Vehicle:

yes

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, and were prepared twice during the study, with the exception of the highest test concentration that was prepared once during the test. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 90 mg a.i./mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 1.1, 3.3, 10 and 30 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and appeared clear and colorless. Stock solutions were stored refrigerated in 500 mL glass amber bottles, and aliquots of each stock were placed in the syringe pump every two days during the study.

The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.5 µL/minute where they were mixed with dilution water delivered at a rate of 155 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.
- Controls: Yes, blanks and vehicle controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 ml/l
- Syringe pumps used to deliver stock solutions or solvent to the mixing chambers were calibrated prior to test. Rotameters used to control flow of dilution water to the mixing chambers were calibrated prior to test and calibrated/verified weekling during the test. Flow rates varied by no more than circa 10 % of the meand flow rate for the two replicates. Delivery of test solutions to test chambers was initiated approx. 45 h prior to introduction of test animals. General operation of exposure system was checked visually at leaste once or twice per day during the test.

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 of parental stock (mean and range, SD): less than 24 hours
- Feeding during test
- Food type: mixture of yeast, cereal grass media and trout chow (YCT), combined vitamin stock (0.2 mL), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Amount: At each feeding during the test, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae, and supplemented with 0.5 mL suspension of combined vitamin stock once daily. This amount of feed is equal to approximately 0.71 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the system to support acceptable reproduction rates.
- Frequency: Daphnids were fed two to four times per day through Day 7 of the test and then were fed four times per day until the last day of the test, except on Day 10 of the test daphnids were fed three times per day.

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions (same as test or not): same
- Type and amount of food: same as during test
- Feeding frequency: same as during test
- Health during acclimation (any mortality observed): no signs of disease or stress observed.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The four adults used to supply neonates for the test were held for at least 13 days prior to collection of the juveniles for testing and had each produced at least one previous brood. They had produced an average of at least 3 young per adult per day over the 7-d period prior to the test. The adults showed no signs of disease or stress or sphippia. For the test, junevils were collected and indiscriminately transferred to transfer chambers until all of them contained 5 daphnids. Each group of neonates was then impartially assigned to a control or treatment group and the neonates were transferred to the test compartment to initiate the test.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

132 - 141 mg/l CaCO3

Test temperature:

19.9 - 20.3 ºC

pH:

8.0 - 8.1

Dissolved oxygen:

Mean oxygen concentrations per exposure concentration: 7.0 - 8.0 mg/l
Concentrations remained above 61% of saturation level (5.5 mg/l) at all times

Nominal and measured concentrations:

Nominal: 0.11, 0.33, 1.0, 3.0 and 9.0 mgl
Mean measured: 0.10, 0.28, 0.83, 2.5 and 7.7 mg/l,
Representing: 91, 85, 83, 83 and 86% of the nominal concentrations, respectively.
Therefore the nominal concentrations acan be used to report the final results.
Individual concentrations at all times ranged from 72 to 104% of nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 ml glass beakers. Nylon mesh covered two holes on opposite sides of each test compartment to permit test solution to flow in and out.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 300 ml. Placed in stainless steel aquaria of 25 liters filled with 22 l of medium
- Aeration: Yes, in storage tank
- Renewal rate of test solution (frequency/flow rate): 5 renewals of test volume per day
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well located 40 meters deep on the Wildlife International Ltd. site. Wat er is passed through a sand-filter, filtered to 0.45 um and passed through a UV-sterilizer.
- Metals: all metals below limit of quantification, ecept for Calcium (34 mg/l), chloride (4.5 mg/l), fluoride (0.85 mg/l), magnesium (13.2 mg/l), potassium (7.0 mg/l), sodium (19.0 mg/l) and sulfate (5.6 mg/l).
- Pesticides: all pesticides below limit of quantification
- Alkalinity: 178 mg/l CaCO3
- Conductivity: 362 microS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement:
Temperature: measured at start of the test, at weekly intervals and at the end of the test
Dissolved oxygen: measured at start of the test, three times a week during the test and at the end of the test
pH: measured at start of the test, at weekly intervals and at the end of the test

OTHER TEST CONDITIONS
- Photoperiod: 16h light / 8h dark, with 30 minute transition period. Light intensity 613 lux at water surface.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.24, 0.81, 2.7, 9.0 and 30 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95%CL: 1.3 - 2.6 mg/l

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95%CL: 1.8 - 4.3 mg/l

Details on results:

- Mortality of parent animals:
5% (negative control),
25% (solvent control)
0% (0.10 mg/l)
5% (0.28 mg/l)
35% (0.83 mg/l)
55% (2.5 mg/l)
100% (7.7 mg/l)
- No. of offspring produced per day per female:
Negative control: 12.9 +/- 0.98
Solvent control: 13.9 +/- 1.8
0.10 mg/l: 13.1 +/- 0.68
0.28 mg/l: 12.4 +/- 0.87
0.83 mg/l: 14.6 +/- 1.8
2.5 mg/l: 6.8 +/- 2.0
7.7 mg/l: -
- Body length and weight of parent animals:
Negative control: 4.8 +/- 0.05 mm; 1.12 +/- 0.07 mg
Solvent control: 4.8 +/- 0.14 mm; 1.20 +/- 0.13 mg
0.10 mg/l: 4.8 +/- 0.15 mm; 1.08 +/- 0.08 mg
0.28 mg/l: 4.7 +/- 0.08 mm; 1.10 +/- 0.08 mg
0.83 mg/l: 4.9 +/- 0.08 mm; 1.22 +/- 0.13 mg
2.5 mg/l: 4.2 +/- 0.32 mm; 1.12 +/- 0.04 mg
7.7 mg/l: -

Reported statistics and error estimates:

Survival data was considered to be discrete-variable data, while reproduction and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the solvent control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the solvent control means were identified using Dunnett’s test (α = 0.05). All statistical tests were performed using a personal computer with TOXSTAT (5) or SAS (6) software.

Measured Concentrations of Cyclaprop in Test Solution Samples 

Nominal Test Concentration (mg/L)	Sample Number (558A-119-)	Sampling Time (Days)	Measured Concentration^1 (mg/L)	Percent of Nominal^2	Mean Measured Concentration (mg/L)	Mean Percent of Nominal
Negative Control (0.0)	1 8 15 21	0 7 14 21	< LOQ < LOQ < LOQ < LOQ	-- -- -- --	--	--
Solvent Control (0.0)	2 9 16 22	0 7 14 21	< LOQ < LOQ < LOQ < LOQ	-- -- -- --	--	--
0.11	3 10 17 23	0 7 14 21	0.103 0.100 0.0982 0.0950	93.8 91.2 89.3 86.4	0.10 (%CV = 3.4)	91
0.33	4 11 18 24	0 7 14 21	0.342 0.279 0.267 0.238	104 84.4 80.9 72.1	0.28 (%CV = 16)	85
1.0	5 12 19 25	0 7 14 21	0.906 0.822 0.843 0.741	90.6 82.2 84.3 74.1	0.83 (%CV = 8.2)	83
3.0	6 13 20 26	0 7 14 21	2.70 2.49 2.48 2.33	90.2 83.1 82.6 77.6	2.5 (%CV = 6.1)	83
9.0	7 14	0 7	8.66 6.80	96.2 75.5	7.7 (%CV = 17)	86
^1   The limit of quantitation (LOQ) was 0.0500 mg/L, calculated as the product of the concentration of the lowest calibration standard (0.100 mg/L) and the dilution factor of the matrix blank samples (0.500). ^2   Results were generated using Excel 2000 in full precision mode. Manual calculations may differ slightly.

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to Cyclaprop for 21 Days

Mean Measured Concentration (mg/L)	Percent Adult Survival^1	Mean No. Neonates Per Reproductive Day ± Std. Dev.^2	Mean Length ± Std. Dev. (mm)	Mean Dry Weight ± Std. Dev.^3 (mg)
Negative Control	95	12.9 ± 0.98	4.8 ± 0.05	1.12 ± 0.07
Solvent Control	75	13.9 ± 1.8	4.8 ± 0.14	1.20 ± 0.13
0.10	100	13.1 ± 0.68	4.8 ± 0.15	1.08 ± 0.08
0.28	95	12.4 ± 0.87	4.7 ± 0.08	1.10 ± 0.08
0.83	65	14.6 ± 1.8	4.9 ± 0.08	1.22 ± 0.13
2.5	45^4	6.8 ± 2.0*	4.2 ± 0.32*	1.12 ± 0.04
7.7	0*^, 5	--	--	--
The concentrations in this table are based on measured concentration. For classification and labelling, PNEC derivation and PBT assessment the nominal concentraitons will be used because the measured concentrations are > 80% of the nominal test concentrations. * Indicates a statistically significant decrease in percent survival (Fisher’s Exact test,p ≤ 0.05), mean total length and reproduction in comparison to the solvent control (using Fisher’s Exact test,p ≤ 0.05 for percent survival and Dunnett’s one-tailed test,p ≤ 0.05 for mean total length and reproduction). ^1  21-day EC50 for survival (95%CI): 1.6 mg/L (1.0 – 2.2 mg/L). ^2  21-day EC50 for reproduction (95% CI): 2.1 mg/L (1.5 – 4.0 mg/L). ^3No statistically significantreduction in mean dry weight was found in any of the treatment concentration in comparison to the solvent control (Dunnett’d one-tailed test, p >0.05). ^4  Although no statistically significant difference was found in comparison to the solvent control, the 65% immobility noted in this treatment group is considered to be biologically significant. 5 Growth and reproduction data from the 7.7 mg/L treatment concentrations were not analyzed for the growth or reproduction endpoints since no adult survived to test end.

The concentration-response curve for immobility of first-generation daphnids exposed to Cyclaprop for 21 days is included as illustration below.

Validity criteria fulfilled:

yes

Remarks:

Parental mortality was 25% in solvent control (exceeding the 20%). In the lowest test concentration no immobility was observed and therefore this deviation is considered not have affected the results;

Conclusions:

The 21d-NOEC reproduction of Cyclaprop to Daphnia magna is at least 1 mg/l. The 21d-EC50 for mobility and for reproduction are 1.8 and 2.6 mg/l, respectively.

Executive summary:

The chronic toxicity of Cyclaprop towards Daphnia magna was investigated according to OECD guideline 211 under GLP. Daphnids were exposed to nominal concentrations of 0.11, 0.33, 1.0, 3.0 and 9.0 mg/l for 21 days in a flow-through system. Mean measured concentrations were within 20% of the nominal concentrations, therefore nominal concentrations were used to calculate the results.

The NOEC and EC50 for survival were 1 and 1.8 mg/l, respectively. The NOEC and EC50 for reproduction were 1 and 2.6 mg/l, respectively.

Some issues with oily slick in the test chambers were seen presenting potentially physical effects. At the test concentrations, however, no differentiation could be made between physical and systemic effects and therefore the above mentioned values are used for classification and labelling, PNEC derivation and PBT assessment.

Reference 2

Endpoint:

long-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 long-term toxicity for Cyclacet Dihydro has been derived from acute toxicity of Cyclacet and the acute / chronic ratio of Cyclaprop. The information is justified in the Endpoint summary on Aquatic toxicity. The accompanying files are also at the Endpoint summary.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Acute / Chronic ratio

Validity criteria fulfilled:

not specified

Executive summary:

Cyclacet Dihydro's long term Daphnia toxicity is derived from acute acute Daphnia toxicity of Cyclacet, which is 25 mg/l and the acute / chronic ratio of Cyclaprop, which is 14. This results in a long-term fish toxicity value of 1.8 mg/l. A full justification is copied into the Endpoint summary.

Description of key information

The chronic toxicity of Daphnia, EC10, is 1.8 mg/l using read across from Cyclacet (OECD TG 202) and acute / chronic ratio of Cyclaprop of Daphnia (OECD TG 202 and OECD TG 211).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.8 mg/L