cis-2-tert-butylcyclohexyl acetate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Mar 2016 to 30 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 2012

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

1996

Qualifier:

according to guideline

Guideline:

other: ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests

Version / remarks:

2004

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from each treatment and control group at test initiation, at the beginning and the end of the longest renewal cycle each week, and at test termination. Samples of “new” solutions were collected from the bulk preparations of each test solution at test initiation and at the beginning of the longest renewal cycle (i.e., on Days 0, 3, 10 and 17 of the test). Samples of “old” solutions were collected from two alternating replicates of each treatment and control group at the end of the longest renewal cycle and at test termination (i.e., on Days 3, 6, 13, 17 and 21 of the test). The samples were collected volumetrically from mid-depth, placed in Teflon™ centrifuged tubes, and processed immediately for analysis.

Vehicle:

yes

Remarks:

0.1 mL HPLC-grade dimethylformamide/L

Details on test solutions:

Test solutions were prepared eight times during the test, every 1 to 3 days (i.e. on Monday, Tuesday, Wednesday and Friday). A primary stock solution was prepared at a nominal concentration of 100 mg/mL in HPLC-grade dimethyformamide (DMF). The primary stock solution was sonicated for 20 to 30 minutes followed by inversion to mix, and appeared clear and colorless. Proportional dilutions of the primary stock solution were made in DMF to prepare secondary stock solutions. The secondary stock solutions appeared clear and colorless. Aliquots of the primary and secondary stock solutions (0.20 mL) were dissolved in 2000 mL of dilution water to prepare test solutions at nominal concentrations. The test solutions were mixed by inversion, and approximately 200-mL aliquots were added to each test chamber. All test solutions appeared clear and colorless with no visible precipitate noted.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnid
- Source: Wildlife International, Easton, Maryland
- Age: Daphnid neonates used in the test were less than 24 hours old
- Feeding: During culturing and testing, daphnids were fed daily a mixture of yeast, cereal grass media and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Raphidocelis subcapitata. At each feeding, each test chamber was fed 0.5 mL of YCT, 1.0 mL of algae and 0.4 mL of vitamin solution. This amount of feed is equal to approximately 0.6 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.

CULTURING CONDITIONS
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 19.5 to 20.8ºC, measured with a hand-held, digital thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥7.9 mg/L (≥88% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter.

BREEDING
The three adult daphnids used to supply neonates for the test were held for at least 14 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. To initiate the test, 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 neonates then was impartially assigned to a control or treatment group and individual neonates were transferred to each test chamber to initiate the test. All transfers were made below the water surface using wide-bore pipettes.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

136 - 152 mg/L as CacO3

Test temperature:

19.2 - 20.9 °C

pH:

7.7 - 8.6

Dissolved oxygen:

6.7 - 9.1 mg O2/L

Conductivity:

334 - 360 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0 and 10 mg/L
- Time-weighted mean measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: The test chambers were 8 oz. glass French square bottles with lids, and contained approximately 200 mL of test solution. The depth of solution in a negative control replicate A test chamber was 8.7 cm. The test chambers were impartially positioned in an environmental chamber to maintain the target water temperature throughout the test period. All test chambers were labeled with the project number, test concentration and replicate designation.
-Type: Due to the volatile nature of the test substance, test chambers were closed bottles with minimal headspace in an attempt to maintain test concentration.
- Renewal rate of test solution: Test solutions were renewed every 2-3 days.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10
- No. of vessels per vehicle control: 10

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 site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, 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 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer. The well water is characterized as moderately-hard water.
- Total organic carbon: Total organic carbon measured in the dilution water at test initiation and termination was <1 mg C/L.
- Metals: Please see 'Any other information on materials and methods incl. tables' for an overview of the metals analysed in the test medium.
- Pesticides: Please see 'Any other information on materials and methods incl. tables' for an overview of the pesticides and organics analysed in the test medium.
- Chlorine: 4 mg/L
- Ca/mg ratio: 2.59
- Alkalinity: 174 - 178 mg/L as CACO3
- Culture medium different from test medium: same

TEST MEDIUM MEASUREMENTS
- Temperature: The target test temperature during the test was 20 ± 1°C. Temperature was measured in two replicate test chambers in each treatment and control group at test initiation, in the old and new solutions on renewal days, and at test termination using a digital thermometer. Measurements typically rotated among the replicates in each group at each measurement interval. Temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (Amegaview Central Monitoring System), which were calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a digital thermometer.
- Dossilved oxygen and pH: Dissolved oxygen and pH were measured in the newly prepared batch solutions for each treatment and control group at test initiation and on renewal days, and in the old solutions from two replicate test chambers in each treatment and control group on renewal days and at test termination. Measurements typically rotated among the replicates in each group at each measurement interval. Dissolved oxygen was measured using a Thermo Orion Star A213 dissolved oxygen meter. Measurements of pH were made using a Thermo Orion Dual Star pH/ISE meter. When a first-generation daphnid was found dead, measurements of temperature, dissolved oxygen and pH were taken in the replicate at that time, and then discontinued.
- Hardness, alkalinity, conductivity: Hardness, alkalinity and specific conductance were measured in batch solutions of the negative (dilution water) control and the highest test concentration at test initiation and on one renewal day each week (Days 8 and 15), and from pooled replicate solutions at test termination. Total organic carbon (TOC) was measured in the dilution water at test initiation and termination. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. Specific conductance was measured using an Acorn Series Model CON6 Conductivity-Temperature meter. TOC was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer based on procedures in Standard Methods for the Examination of Water and Wastewater.

OTHER CONDITIONS
- Illumination: Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight.
- Light intensity: Light intensity at test initiation was 1428 lux. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.
- Photoperiod: 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.

EFFECT PARAMETERS MEASURED: parental immobility, reproduction, body length and weight offspring
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, lethargy, opaque color or cessation of feeding). Those daphnids that are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobilized (even if they can still move their antennae). The presence of eggs in the brood pouch, aborted eggs, males or ephippia also was 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 and at test termination. Second-generation daphnids were also counted at the time a replicate was terminated due to a first-generation immobility. The body length and dry weight of each surviving first-generation daphnid were measured at the end of the test.

TEST CONCENTRATIONS
- Range finding studies: In order to select the most appropriate test design for the test material, two non-GLP range-finding tests were conducted under static-renewal conditions and flow-through conditions for 14 days. The range finding tests were conducted at nominal concentrations of 0.081, 0.27, 0.90, 3.0 and 10 mg/L along with a negative control and solvent control (0.10 mL HPLC-grade dimethyformamide/L).
- Results used to determine the conditions for the definitive study: The mean number of live neonates produced per reproductive day in the negative control, solvent control, 0.081, 0.27, 0.90, 3.0 and 10 mg/L treatment groups for the semi-static range-finding test was 131, 125, 113, 142, 130, 117 and 65.3, respectively. For the flow-through range-finding test this number was 11.8, 10.0, 11.0,
11.4, 13.9, 12.9 and 3.7, respectively. An apparent impact on reproduction was noted in the results from both range finding tests. The nominal test concentrations were selected based on these results.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.99 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

per parent animal which does not die accidentally or inadvertently during the test

Remarks on result:

other: 95% C.I.: 0.76 - 1.3 mg/L

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

per surviving parental animal at the end of the test

Remarks on result:

other: 95% C.I.: 0.78 - 1.4 mg/L

Details on results:

A summary of the survival, clinical observations and reproduction of the test is presented in 'Any other information on results incl. tables'.
- Mortality: Survival in the pooled control, 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L treatment groups at test termination was 100, 100, 90, 80, 60 and 10%, respectively.
- Sub-lethal effects: Daphnids in the 0.17, 0.39, 0.91 and 1.9 mg/L treatment groups that survived to test termination generally appeared normal. Transient observations of discoloration was noted for three daphnids in the 1.9 mg/L treatment group, but all surviving daphnids in this group were normal in appearance at test termination. In the 3.5 mg/L treatment group, clinical signs of toxicity included lethargy, discoloration, erratic swimming and/or small in stature from Day 8 through Day 21 of the test. Only one daphnid in the 3.5 mg/L treatment group survived to the end of the test. The clinical signs of toxicity noted in the 3.5 mg/L treatment level were considered to be treatment related.
- Reproduction: There was no apparent delay win the onset of production at any treatment. With the exception of one and 9 immobile neonates in the solvent control and the 3.5 mg/L treatment, respectively, on Day 13 of the test, no immobile neonates were noted in the negative control or the treatment groups during the test. No aborted/shed eggs, males or ephippia were produced during the test. Adult daphnids in the negative control and solvent control groups produced an average of 283 and 280 live young per surviving adult, respectively. Adult daphnids in the 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L treatment groups produced an average of 270, 269, 247, 180 and 60 live young per surviving adult, respectively.
- Growth: Daphnids in the negative control group averaged 4.80 mm in length and 0.84 mg in dry weight, while daphnids in the solvent control group averaged 4.87 mm in length and 0.98 mg in dry weight. There were no statistically significant differences in mean total length or dry weight between the negative and solvent control, the control data were pooled for comparison with the treatment data. Daphnids in the pooled control and the 0.17, 0.39, 0.91 and 1.9 mg/L treatment groups had mean lengths of 4.84, 4.82, 4.68, 4.68 and 4.4 mm, respectively, and mean dry weights of 0.91, 0.97, 0.83, 0.99 and 1.16 mg, respectively. There was only one remaining daphnid in the 3.5 mg/L treatment level and it was 3.4 mm in length and 0.59 mg in dry weight.

Reported statistics and error estimates:

Please see 'Any other information on materials and methods incl. tables' for a detailed description of the statistical analyses of the study.

Table: Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to the test item for 21 Days

Time-Weighted Mean Measured Concentration (mg/L)	Percent Adult Survival	Mean No. Neonates Per Surviving Adult (# Surviving Adults) ± Std. Dev.	Mean No. Neonates Per Adult At the Start of the Test ± Std. Dev.	Mean Length ± Std. Dev. (mm)	Mean Dry Weight ± Std. Dev. (mg)(b)
Negative control	100	283 ± 50 (10) (%CV = 18)	283 ± 50 (%CV = 18)	4.80 ± 0.16	0.84 ± 0.16
Solvent Control(b)	100	280 ± 44 (10)	280 ± 44	4.87 ± 0.15	0.98 ± 0.28
Pooled Control	100	282 ± 46	282 ± 46	4.84 ± 0.16	0.91 ± 0.23
0.17	100	270 ± 19 (10)	270 ± 19	4.82 ± 0.11	0.97 ± 0.23
0.39	90	269 ± 20 (9)	257 ± 44	4.68 ± 0.14*(c)	0.83 ± 0.10
0.91	80	247 ± 25* (8)	227 ± 46*	4.68 ± 0.14*(c)	0.99 ± 0.27
1.9	60*	180 ± 44* (6)	134 ± 74*	4.40 ± 0.18*(c)	1.16 ± 0.19
3.5	10*	60(a) (1)	21.2 ± 23*	3.40(a)	0.59(a)
EC10 & 95% CI (mg/L)	NA	1.0 (0.78 – 1.4)	0.99 (0.76 – 1.3)	NC	NC
EC50 & 95% CI (mg/L)	1.7 (1.1 – 2.9)	2.3 (2.0 – 2.7)	1.8 (1.6 – 2.1)	NC	NC

* Indicates a significant differences from the pooled control in survival (Fisher’s Exact test, p≤0.05), in total length, in reproduction (mean number of neonates

per surviving adult at test end) (Dunnett’s one-tailed test, p≤0.05) and in reproduction (mean number of neonates per adult at the beginning of the test

(Bonferroni’s t-test, p≤0.05).

a) No standard deviation was calculated since there was only one value. The reproduction (measured as mean number of live neonates produced per surviving adult) and growth data of the 3.5 mg/L treatment group were excluded from the statistical analysis because there was only one daphnid remaining in the treatment group at test termination.

b) There were no statistically significant reductions in dry weight in comparison to the pooled control (Dunnett’s one tailed test, p>0.05).

c) Because the differences in mean total length of the daphnids in the 0.39, 0.91 and 2.0 mg/L treatment groups were slight and < 10% (3.3, 3.3 and 9.1%, respectively), the differences were not considered to be biologically meaningful.

NA = not applicable; NC = not calculated

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl tables'.

Conclusions:

The 21-d EC10 for reproduction is 0.99 mg/L in freshwater invertebrates (D. magna) in an OECD T 211 test.

Executive summary:

The 21-day chronic toxicity to aquatic invertebrates was determined in a study according to OECD 211 and in compliance with GLP criteria. In this study daphnids (D. magna, 10 per concentration) were exposed to nominal concentrations of 0 (control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0 and 10 mg/L for 21 days under semi-static conditions. Analytical confirmation of test concentrations showed that the test concentrations did not remain within ±20% of nominal concentrations throughout the test. Therefore, the calculated time-weighted mean measured concentrations of <LOQ (control), <LOQ (solvent control), 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L were used for the derivation of the effect values. First-generation daphnids were observed daily during the test for immobility, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week and at test termination. Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period. The water quality parameters measured at test initiation and on renewal days, and in the old solutions from two replicate test chambers in each treatment and control group on renewal days and at test termination were determined to be within acceptable limits. The validity criteria of the test guidelines were fulfilled. Survival in the pooled control, 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L treatment groups at test termination was 100, 100, 90, 80, 60 and 10%, respectively, resulting in an EC50 mortality of 1.7 mg/l. Length and dry weight were not considered for long-term toxicity because effects were <10%, at the concentrations where no mortality was seen. Mean neonate production per surviving adult significantly decreased in the three highest test concentrations in a dose dependent manner, in comparison with the pooled control (270, 269, 247, 180 and 60, respectively), resulting in a NOEC of 0.39 mg/l, EC10 of 1 mg/l. The EC10 of 0.99 for the reproduction using all parental daphnids is used as the key value.

Description of key information

The 21-day chronic toxicity to aquatic invertebrates was determined in a study according to OECD 211 and in compliance with GLP criteria. In this study daphnids (D. magna, 10 per concentration) were exposed to nominal concentrations of 0 (control), 0 (solvent control), 0.63, 1.3, 2.5, 5.0 and 10 mg/L for 21 days under semi-static conditions. Analytical confirmation of test concentrations showed that the test concentrations did not remain within ±20% of nominal concentrations throughout the test. Therefore, the calculated time-weighted mean measured concentrations of <LOQ (control), <LOQ (solvent control), 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L were used for the derivation of the effect values. First-generation daphnids were observed daily during the test for immobility, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week and at test termination. Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period. The water quality parameters measured at test initiation and on renewal days, and in the old solutions from two replicate test chambers in each treatment and control group on renewal days and at test termination were determined to be within acceptable limits. The validity criteria of the test guidelines were fulfilled. Survival in the pooled control, 0.17, 0.39, 0.91, 1.9 and 3.5 mg/L treatment groups at test termination was 100, 100, 90, 80, 60 and 10%, respectively, resulting in an EC50 mortality of 1.7 mg/l. Length and dry weight were not considered for long-term toxicity because effects were <10%, at the concentrations where no mortality was seen. Mean neonate production per surviving adult significantly decreased in the three highest test concentrations in a dose dependent manner, in comparison with the pooled control (270, 269, 247, 180 and 60, respectively), resulting in a NOEC of 0.39 mg/l, EC10 of 1 mg/l. The EC10 of 0.99 for the reproduction using all parental daphnids is used as the key value.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.99 mg/L