Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

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:
November 2, 2012 - November 14, 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Settling time for the test solution was up to 90 minutes instead of 60 per the protocol, but the deviation had no adverse impact on results or interpretation of the study.
Principles of method if other than guideline:
Daphnids were exposed to a geometric series of four water-accommodated fraction (WAF) loading rates and a negative (dilution water) control. Ten replicate test chambers containing one daphnid each were tested for each treatment group and 20 replicate test chambers were tested for the control group. Nominal test concentrations were selected in consultation with the Sponsor. The nominal WAF loading rates were 0.01, 0.10, 1.0 and 10 mg C10-13 LAB/L. Test solutions were renewed every 2-3 days. Samples of test water were collected from each treatment and control group at test initiation, at the beginning and end of the longest renewal cycle each week, and at test termination. The samples were processed for analysis by the Sponsor. The results of the study were based on the nominal WAF loading concentrations.
GLP compliance:
yes (incl. certificate)
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. Samples of “old” solutions were collected from alternating replicates of each treatment and the control group at the end of the renewal cycle and at test termination. The samples were collected from mid-depth, placed in glass vials with 20 μL of sodium azide solution (1% w/v) and stored under refrigerated conditions until analyzed.
Details on test solutions:
WAF solutions at nominal WAF loading rates of 0.01, 0.10, 1.0 and 10 mg/L were prepared every 2 to 3 days (typically each Monday, Wednesday and Friday) during the test. Each WAF solution was prepared individually in a glass aspirator bottle by mixing a calculated amount of the test substance into dilution water using a glass-lined magnetic stir bar and stir plate. Care was taken to maintain a vortex depth of approximately 10% of the test solution height during mixing. Following the mixing period of a maximum of 49 hours, the WAF solution was allowed to settle for approximately 60 to 90 minutes and the test solution was decanted. The test solution was decanted into the test chambers (250 mL glass beakers) at the beginning of the test and prior to transferring of the surviving first-generation daphnids into the new solution at each renewal period. Negative control solution was prepared in the same manner with dilution water only.

DETAILS

For the preparation of the WAF solutions the following EtOH stock solutions were used:

- LAB, C10-13 stock solution A, 10k ppm

- LAB, C10-13 stock solution B, 1k ppm 1 mL LAB stock solution A in 10 mL EtOH


The WAF (working) solutions, containing 1 ppm of n-decanol as internal standard, at the nominal given concentration were prepared as follows:

- LAB, C10-13 (4 ppm): 200 uL LAB stock solution A + 50 uL internal standard stock solution in 500 mL H2O (MilliQ)

- LAB, C10-13 (1 ppm): 50 uL LAB stock solution A + 50 uL internal standard stock solution in 500 mL H2O (MilliQ)

- LAB, C10-13 (4 ppm): 200 uL LAB stock solution B + 50 uL internal standard stock solution in 500 mL H2O (MilliQ)

- LAB, C10-13 (1 ppm): 50 uL LAB stock solution B + 50 uL internal standard stock solution in 500 mL H2O (MilliQ)

These solutions were stirred for 12 hours at 23 degrees celsius (TeflonTM coated magnetic bar), settled for 24 hours and then an aliquot was taken from the bottom half of the liquid to proceed to the SPME analyses.
Test organisms (species):
Daphnia magna
Details on test organisms:
The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use in the laboratory. Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by Wildlife International, Easton, Maryland.

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 19.4 to 20.7ºC, measured with a hand-held, liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, measured with a Thermo Orion Model 525Aplus pH meter. Dissolved oxygen concentrations were >=7.7 mg/L (>=86% of saturation), measured with a Thermo Orion Model 850Aplus dissolved oxygen meter.

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, Pseudokirchneriella subcapitata. At each feeding, each test chamber was fed 0.5 mL of
YCT, 1.0 mL of algae and 0.40 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.

The three adult daphnids used to supply neonates for the test were held for 17 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:
Negative Control - 143 mg/L CaCO3 (+/- 4)

Highest WAF Loading Rate (10 mg/L) - 142 mg/L CaCO3 (+/- 5)
Test temperature:
Negative Control - 19.9 oC (+/- 0.46)

Highest WAF Loading Rate (10 mg/L) - 20 oC (+/- 0.46)
pH:
Negative Control - 8.2 (+/- 0.07)

Highest WAF Loading Rate (10 mg/L) - 8.3 (+/- 0.1)
Dissolved oxygen:
Negative Control - 7.6 mg/L (+/- 0.62)

Highest WAF Loading Rate (10 mg/L) - 7.8 mg/L (+/- 0.5)
Nominal and measured concentrations:
Nominal WAF Loading Rate:

Negative Control
0.01 mg/L
0.10 mg/L
1.0 mg/L
10.0 mg/L
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
> 10 mg/L
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 10 mg/L
Basis for effect:
reproduction
Details on results:
Survival and Clinical Observations:

After 21 days of exposure, survival in the negative control group was 95%. Survival in the 0.01, 0.10, 1.0 and 10 mg/L WAF loading rates at test termination was 100, 80, 100 and 90%, respectively. Fisher’s Exact test indicated that there were no statistically significant decrease in survival in any of the LAB, C10-13 treatment levels in comparison to the negative control (p > 0.05). Consequently, the NOEL for survival was 10 mg/L nominal WAF loading rate and the LOEL was >10 mg/L, the highest nominal WAF loading rate tested. Based on the mortalities observed in the treatment groups, the 21-day EL50 value was >10 mg/L, the highest nominal WAF loading rate tested.

Daphnids in the 0.01, 0.10, 1.0 and 10 mg/L WAF loading rates that survived to test termination generally appeared normal. Signs of sublethal toxicity including lethargy and discoloration (pale) were noted in one daphnids in the negative control on Day 3 of the test but no other sublethal signs of toxicity were noted in any other daphnid in the control or LAB, C10-13 treatment groups during the test.

Reproduction:

The first day of brood production in the negative control replicates and in all LAB, C10-13 treatment replicates was Day 7, 8 or 9 of the test, indicating that there was no apparent delay in the onset of production at any LAB, C10-13 concentration tested. With the exception of one immobile neonate in the negative control and the 1.0 mg/L nominal WAF loading rate on Day 12 of the test, no immobile neonates were noted in any other treatment groups during the test (Appendix 10). Adult daphnids in the negative control group produced an average of 270 live young per surviving adult (CV = 8.5%). Adult daphnids in the 0.01, 0.10, 1.0 and 10 mg/L WAF loading rates produced an average of 281, 306, 274 and 303 live young per surviving adult, respectively. Bonferroni’s t-test indicated there were no statistically significant decreases in mean neonate production in any of the LAB, C10-13 treatment levels in comparison to the negative control (p > 0.05). Consequently, the NOELR for reproduction was 10 mg/L nominal WAF loading rate and the LOELR was >10 mg/L, the highest nominal WAF loading rate tested. Based on the reproduction observed in the treatment groups, the 21-day EL50 value was >10 mg/L, the highest WAF loading rate tested.

Growth:

Daphnids in the negative control group averaged 4.8 mm in length and 1.11 mg in dry weight. With the exception of the daphnids in Replicate F and J of the 10 mg/L treatment level, all daphnids in the control and treatment groups were noted with eggs in brood. Since the presence of eggs in brood pouches resulting in a slightly elevated dry weight of the daphnid, the decision was made to exclude the dry weights of the daphnids in Replicate F and J of the 10 mg/L treatment level from the calculation of the mean dry weight of the treatment level. Daphnids in the 0.01, 0.10, 1.0 and 10 mg/L treatment groups had mean lengths of 4.6, 4.8, 4.6 and 4.8 mm, respectively. Consequently, the mean dry weights of daphnids in the 0.01, 0.10, 1.0 and 10 mg/L treatment groups was 1.13, 1.19, 1.11 and 0.99 mg, respectively. Dunnett’s test indicated there were no statistically significant decreases in length or dry weight in any of the treatment groups in comparison to the negative control (p > 0.05). Consequently, the NOELR for growth was 10 mg/L, and the LOELR was >10 mg/L, the highest nominal WAF loading rate tested.
Conclusions:
The cladoceran, Daphnia magna, was exposed to LAB, C10-13 at nominal WAF loading rates of 0.01 to 10 mg/L under semi-static conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤10 mg/L, the highest nominal WAF loading rate tested. Consequently, the NOELR was 10 mg/L. The LOELR was >10 mg/L and the Maximum Acceptable Toxicant Level (MATL) was calculated to be >10 mg/L. The 21-day EL50 value for adult immobility and reproduction were both >10 mg/L, the highest nominal WAF loading rate tested.
Executive summary:

This study was conducted by Wildlife International for ECOSOL at the Wildlife International aquatic toxicology facility in Easton, Maryland. An initial trial was conducted from November 7 to 28, 2012 but was terminated due to unacceptable control performance. The in-life phase of the definitive test was conducted from February 22 to March 15, 2013, with dry weight measurements completed on March 18, 2013.

The objective of this study was to determine the effects of LAB, C10 -13 on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under semi-static test conditions. The test organisms were exposed to LAB, C10-13 at nominal WAF loading rates of 0.01 to 10 mg/L under semi-static conditions for 21 days. There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤10 mg/L, the highest nominal WAF loading rate tested. Consequently, the NOELR was 10 mg/L. The LOELR was >10 mg/L and the Maximum Acceptable Toxicant Level (MATL) was calculated to be >10 mg/L. The 21-day EL50 value for adult immobility and reproduction were both >10 mg/L, the highest nominal WAF loading rate tested.

Description of key information

LAB is the most water soluble component of the HABs. There are two studies determining the chronic toxicity of LAB to aquatic invertebrates.

The key study examined the effects of chronic exposure of Daphnia magna to the test substance, using the water accommodated fraction (WAF) method (OECD Guideline 211). Ten first-instar Daphnia (<24 h old) were individually placed in test chambers with test solution (200 mL) containing 0, 0.01, 0.1, 1.0 or 10.0 mg/L (WAF) of the test substance. Test solutions were changed every 2-3 days. Effect parameters measured included mortality, onset of reproduction (measured three times per week) signs of toxicity, body length and dry weight. Due to the low water solubility of the test substance, no test substance was detected in the analytical monitoring (SPME-GC-FID). The results are therefore reported as a WAF (nominal concentration). The 21-day EL50 for Daphnia magna exposed to LAB was >10 mg/L (WAF) for both immobilization and reproduction. The 21-day NOEL for Daphnia magna was 10 mg/L (WAF) and the 21-day LOEL was >10 mg/L (WAF). Based on these results LAB is not classified for chronic aquatic toxicity.

In the second study, groups of 10 daphnids were exposed to concentrations of 1.9, 3.8, 7.5, 15 or 30 μg/L of test substance for 21 days. The tests were conducted in a flow-through proportional diluter system, with a solvent (1 ml/L acetone). There were ten first-instar Daphnia (< 24h old) in each of the four replicate chambers for each test substance concentration, the control and the solvent control. Survival and reproduction were recorded and assessed three times a week. Growth was established only at day 21 by measuring mature daphnids. Biological parameters included adult survival, total young produced and young produced per adult and were summarized on days 0, 4, 7, 14 and 21. Reproduction and growth was reduced at concentrations between 15-30 μg/l. The 21-day NOEC for daphnids was 0.0075 mg/L. The 21-day LOEC was 0.015 mg/L. The 21-day EC50 for daphnids was 0.012 mg/L. The results can be considered conservative as the solvent concentration (1 mL/L) was above the currently recommended levels for solvent addition (<0.1 mL/L) (OECD 2000, OECD 2008).

Key value for chemical safety assessment

Additional information