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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-11 to 2020-01-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 batch preparations of each test solution at test initiation and at the beginning of the longest renewal cycle. 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. The samples (8.0 mL) were collected from mid-depth, placed in glass scintillation vials containing 2.0 mL acetonitrile and processed immediately for analysis.
Vehicle:
no
Details on test solutions:
Dilution Water
The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Eurofins-Easton 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. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) content of the well water during the approximate four-week period immediately preceding the test are presented in Appendix 4 (cf. attached background information).

Preparation of Test Concentrations
Test solutions were prepared every 1 to 3 days (e.g., on Monday, Wednesday and Friday) during the test. At each preparation, two primary stock solutions at concentrations of 5.0 and 10 mg/L were prepared. Calculated amounts of test substance (10.0 mg) were weighed into weigh boats and the test substance were rinsed into 1000 and 2000 mL glass volumetric flasks using approximately 30 mL of HPLC-grade acetone. Once all test substance was transferred into the flasks, acetone was allowed to evaporate for approximately 30 to 60 minutes. Then the flasks were brought to volume using reversed osmosis (RO) water and the solutions were stirred overnight to mix using Teflon®-lined stirred bars and magnetic stir plates. The stock solutions appeared clear and colorless with small amount of material stuck to the side of the flasks or clear and colorless in the 5.0 mg/L stock concentration and clear and colorless with small amount of white precipitate on the bottom of the flask of the 10 mg/L stock concentration.
Aliquots of the 5.0 and 10 mg/L stock solutions were diluted in the dilution water (UV sterilized well water) to prepare test solutions at concentrations of 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L. The test solutions were stirred using Teflon®-lined stirred bars and magnetic stir plates for 30 to 45 minutes. All solutions appeared clear and colorless after mixing. Approximately 200-mL aliquots of test solutions were added to each test chamber at the beginning of the test and at each renewal period, resulting in a loading of >50 mL of test solution per parental generation daphnid.

Test organisms (species):
Daphnia magna
Details on test organisms:
Test Organism
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 Eurofins, Easton, Maryland. Identification of the species was verified by the supplier of the original stock culture.
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 20.0 to 20.8ºC, measured with a digital thermometer. The pH of the water ranged from 8.0 to 8.7, measured with a Thermo Scientific Orion Benchtop 4 StarPlus pH/ISE meter. Dissolved oxygen concentrations were =7.5 mg/L (=83% of saturation), measured with a Thermo Scientific Orion Benchtop 3 Star Plus 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 suspension of the freshwater green alga, Raphidocelis subcapitata and a vitamin stock solution. At each feeding during the test, each test chamber was fed 0.5 mL of YCT, and 1.0 mL of algae. In addition, each test chamber also received 0.40 mL of vitamin solution once daily. This amount of feed is equal to approximately 0.60 mg C/daphnid/day based on a non-GLP test. 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 19 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 5 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:
116 - 144 mg/L as CaCO3
Test temperature:
19.7 - 20.7 °C
pH:
7.9 - 8.6
Dissolved oxygen:
7.0 - 9.1 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L
Measured concentrations: cf. "any other information on results incl. tables".
Details on test conditions:
Environmental Conditions
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 840006 light meter.
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 and in two replicate test chambers in the old and new solutions on renewal days, and in the old solutions at test termination using a digital thermometer. Measurements typically rotated among the replicates in each group of old solutions at each measurement interval. Temperature also was monitored continuously in a container of water placed adjacent to the test chambers using a validated environmental monitoring system (Pointview Central Monitoring System), which were calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a digital thermometer.
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 of old solutions at each measurement interval. Dissolved oxygen was measured using a Thermo Scientific Orion Star A213 bench top RDO/DO meter. Measurements of pH were made using a Thermo Scientific 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 and specific conductance were measured in batch solutions of the negative (dilution water) control and the highest test concentration at test initiation (Day 0) and on one renewal day each week (Days 8 and 15), and from pooled replicate solutions at test termination (Day 21). 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 a Thermo Orion Star A122 portable conductivity meter.

Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.51 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Adult survival, production rate of first brood, mean no. neonates per adult/per surviving adult, length, dry weight
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 0.51 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Adult survival, production rate of first brood, mean no. neonates per adult/per surviving adult, length, dry weight
Details on results:
See "any other information on results incl. tables".

Measurement of Test Concentrations

Nominal concentrations selected for use in the study were 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L. Test solutions in the test chambers at these nominal concentrations appeared clear and colorless during the test, and clear and light green in color due to algal feed at the end of the renewal periods and at test termination, with no evidence of precipitation observed. Results of analyses to measure concentrations of BADGEDA in water samples collected during the test are presented in Table 1 (cf. attached files - background information). Concentrations of BADGEDA in the new test solutions prepared and sampled on Days 0, 3, 10, 17 and 20 ranged from 45.9 to 84.7% of the nominal concentrations. Concentrations of BADGEDA in the old test solutions sampled immediately prior to renewal on Days 1, 6, 13, 20 and at termination on Day 21 ranged from <LOQ to 71.6% of the nominal concentrations. The measured concentrations of the samples collected during the test were averaged for each treatment group using time-weighted average, and were 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L, which represented 58, 56, 56, 57 and 51% of the nominal concentrations, respectively. The results of the study are based on the time-weighted mean measured test concentrations.

Physical and Chemical Measurements of Water

Measurements of temperature, dissolved oxygen and pH in the test chambers are summarized in Table 2 (cf. attached files - background information). Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained = 71% of saturation (6.5 mg/L). Measurements of pH ranged from 7.9 to 8.6 during the test. Weekly measurements of specific conductance, hardness and alkalinity in the negative control water and in the highest test concentration are summarized in Table 2. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by BADGEDA concentration. TOC in the dilution water measured approximately monthly was < 1 mg C/L (Table 2). Light intensity at test initiation was 565 lux at the surface of the water of one representative test chamber.

Survival and Clinical Observations

A summary of survival among the individually-exposed first-generation daphnids is presented in Table 3 (cf. attached files - background information). After 21 days of exposure, survival in the negative control was 90%. Survival in the 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L treatment groups at test termination was 90, 100, 50, 80 and 90%, respectively. A statistically significant reduction in percent survival was noted in the 0.14 mg/L treatment group when compared to the negative control (Fisher’s Exact test, p = 0.05). However, the percent survival noted in this treatment group did not follow a dose response pattern and was not considered to be biologically meaningful. Consequently, the NOEC for survival was 0.51 mg/L and the LOEC was > 0.51 mg/L. Based on the immobility observed in the treatment groups, the 21-day EC10, EC20 and EC50 values based on the percent survival observed were estimated to be > 0.51 mg/L, the highest concentration tested (Table 3). The percent inhibition in percent survival in each treatment concentration compared to the negative control is shown in Table 4 (cf. attached files - background information).

Daphnids in the 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L treatment groups that survived to test termination generally appeared normal. There were occasional observations of small and/or discolored (e.g., pale) daphnids during the test. Since the occurrence was infrequent, it was not considered to be treatment related.

Reproduction

A summary of neonate production by surviving first-generation daphnids is presented in Table 3. The first day of brood production in all replicates of the treatment and control groups was on Day 8 of the test, with the exception of a delay noted in the 0.25 and 0.50 mg/L treatment groups. The first day of brood production in the 0.25 mg/L treatment group was on Day 10 in Replicates C and D and on Day 15 in replicate G, while the first day of brood production in replicate C of the 0.50 mg/L treatment group was on Day 10 of the test. The mean production rate of the first brood in the negative control, 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L treatment groups was 0.13, 0.13, 0.13, 0.12, 0.13 and 0.13 per day. No statistically significant difference in production rate of first brood was noted in any of the treatment from the negative control (Jonckheere-Terpstra step down trend test, p > 0.05). Consequently, the NOEC and LOEC based on the production rate of first brood was 0.51 and >0.51 mg/L treatment groups, respectively.

There were total of 1, 48 and 44 immobile neonates produced during the test in the negative control, 0.14 and 0.29 mg/L treatment groups, respectively. There were total of 5, 33, 1 and 2 aborted/shed eggs in the 0.037, 0.14, 0.29 and 0.51 mg/L treatment groups during the test. The productions of immobile neonates and aborted/shed eggs did not follow a dose response pattern and were not considered to be treatment related. No males or ephippia were produced during the test.

Adult daphnids in the negative control group produced an average of 233 live young per surviving adult (CV = 11.5%). Adult daphnids in the 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L treatment groups produced an average of 253, 224, 217, 212 and 275 live young per surviving adult, respectively. There were no statistically significant decreases in mean neonate production per surviving adult in any of the BADGEDA treatment groups in comparison to the negative control (Dunnett’s one-tailed test, p > 0.05).

Adult daphnids in the negative control group produced an average of 227 live young per adult at the beginning of the test (CV = 14.2%). The mean number of neonates produced per adult at the beginning of the test in the 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L treatment groups was 240, 224, 162, 173 and 276, respectively. No statistically significant decreases in mean neonate production per adult at the beginning of the test in any of the BADGEDA treatment groups in comparison to the negative control (Jonckheere-Terpstra step down trend test, p > 0.05).

Consequently, the NOEC for reproduction (mean number of live neonates produced per surviving adult at test end and mean number of live neonates produced per adult at the beginning of the test) was 0.51 mg/L, and the LOEC was > 0.51 mg/L. The 21-day IC10 and IC20 values for reproduction (mean number of neonates produced per surviving adult at test end and mean number of live neonates produced per adult at the beginning of the test) were empirically estimated to be > 0.51 mg/L, the highest concentration tested (Table 3). The percent inhibition in reproduction in each treatment concentration compared to the negative control is shown in Table 4.

Growth

Summaries of the mean lengths and dry weights of surviving first-generation daphnids are presented in Table 3. Daphnids in the negative control and the 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L averaged 4.5, 4.6, 4.5, 4.6, 4.5 and 4.6 mm in length, respectively, and 1.4, 1.5, 1.5, 1.5 and 1.5 mg in dry weight, respectively. No statistically significant reductions in length or dry weight were noted in any of the BADGEDA treatment groups from the negative control (Dunnett’s one-tailed test, p > 0.05 for length and Jonckheere-Terpstra step down trend test, p > 0.05, for dry weight). Consequently, the NOEC and LOEC for growth was 0.51 and >0.51 mg/L, respectively. The IC10 and IC20 for growth were empirically estimated to be > 0.51 mg/L, the highest concentration tested (Table 3). The percent inhibition in growth endpoint in each treatment concentration compared to the negative control is shown in Table 4.

Conditions for the Validity of the Test

The following criteria were used to judge the validity of the test:

1. the percentage of parent daphnids in the negative control that were immobilized and/or appeared to be stressed or diseased at the end of the test will be =20% (it was 10%);

2. the mean number of living offspring produced per parent control animal surviving at the end of the test will be =60 (it was 233 in the negative control);

3. no ephippia were produced by control animals; and

4. among replicate test chambers of a treatment concentration, measured concentrations of the test substance did not vary more than 20% (the %CV ranged from 2.7 to 5.1%).

Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to BADGEDA at mean measured concentrations of 0.037, 0.073, 0.14, 0.29 and 0.51 mg/L under semi-static conditions for 21 days. There were no biologically significant treatment-related effects on reproduction rate of first brood, survival, reproduction (measured as mean number of live neonates produced per surviving adult and mean number of live neonates produced per adult at the beginning of the test) and growth (measured as length and dry weight) at concentrations = 0.51 mg/L. Consequently, the NOEC and LOEC, for the study was 0.51 mg/L and > 0.51 mg/L. The 21-day EC10, EC20 and EC50, values for adult immobility and the IC10 and IC20 values for growth and reproduction (mean number of neonates produced per surviving adult at test end and per adult at the beginning of the test) were > 0.51 mg/L, the highest concentration tested.

Executive summary:

This study was designed to determine the effects of the test item on Daphnia magnareproduction and survival in a 21 days test according to the OECD 211 Guideline (2012).

 

The total test period was 21 days. The test solutions (including control) were renewed every two to three days throughout the test. Twenty replicate test vessels were prepared for the control and ten replicates for each test item concentration (nominal concentrations: 0, 0.063, 0.13, 0.25, 0.50, 1.0 mg/L). A single juvenile Daphnia magna(<24 hours old) was added to each test vessel. On each renewal occasion, parental animals were transferred into freshly prepared test medium. Any juveniles remaining in old test media were counted and the number of juveniles produced per parental animal on any day during the 21-day study duration was recorded.

 

The chemical analysis of test samples showed an instability of the substance between two medium renewals. Based on these results, the exposure concentration was based on the time-weighted average of measured concentrations (TWA measured concentrations: < LOQ (control), 0.037, 0.073, 0.14, 0.29, 0.51 mg/L).

 

The 21 Day EC10 and EC50 of the test item for reproduction, survival and growth in D. magna were concluded to be greater than the highest tested concentration under the test conditions (time weighted average of measured concentrations: 0.51 mg/L). The NOEC was also determined to be equal to or greater than the highest tested concentration.

 

Description of key information

The chronic toxicity of the test substance to Daphnia magna was determined in a semi-static test performed in accordance with the OECD 211 test guideline and GLP requirements.

No significant effects on survival, growth or reproduction were detected up to the highest tested concentration. The 21d-NOEC was equal to or greater than 0.51 mg/L (time-weighted average of measured concentrations). The 21d-EC10 was greater han 0.51 mg/L.

Key value for chemical safety assessment

Additional information