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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 May - 12 Jun 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with analytical monitoring
Qualifier:
according to guideline
Guideline:
other: OECD 202, part 2 adopted in 1984
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2 mg/L treatment
- Sampling method: A separate test concentration was prepared and used for testing the stability of the test substance in water.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by by mixing 1 g test substance with 1 g vehicle in 1 L of dionized water. The stock solution was diluted to the respective test concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): MARLOWET R 40
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 2 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not stated
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: own breeding
- Age of parental stock (mean and range, SD): < 24 hours
- Feeding during test
- Food type: green algae
- Amount: 0.5E7 cells/daphnid from day 0 to day 6; 1.0E7 cells/daphnid from day 7 to day 21
- Frequency: daily

HOUSING
- Housing medium: M4-medium (Elendt, 1990)
- Housing conditions: each test organism were kept separate in 60 mL test water
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
20 ± 1 °C
pH:
7.8 - 8.6
Dissolved oxygen:
94 - 107 mg/L
Nominal and measured concentrations:
Nominal test concentrations: 0.5, 1.0, 2.0 mg/L
Measured concentrations: 0.19, 0.39, 0.77 mg/L (geometric mean, biological active)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, total volume, fill volume: glass beakers, 150 mL, 100 mL
- Renewal rate of test solution (frequency): after 2, 6, 7, 9, 12, 14, 16, 19 days
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic freshwater according to Elendt (M4-medium)
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value and oxygen content of the new test solutions at day 2, 12, 16 and of the old test solutions at day 6, 14, 19.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: approx. 450 and 95 Lux

EFFECT PARAMETERS MEASURED: Immobility and reproduction was recorded after 2, 6, 7 9, 12, 14, 16, 19, 21 days.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Details on results:
No significant effects were observed in up to the highest concentration tested.

Table 1: Results of test substance analytics

Nominal concentration [mg/L]

Determination

Fresh test solution

Old test solution (2 days)

geometric mean [mg/L]

2.0

1

1.8

< 0.05

-

2

1.8

0.86

-

3

1.9

0.12

-

mean

1.8

0.33

0.77

Validity criteria fulfilled:
yes
Conclusions:
A 21d long-term toxicity test with Daphnia magna revealed that DEHA does not cause adverse effects to reproduction and survival up to the saturation limit in water.
Executive summary:

The GLP study was performed according to OECD guideline 202, part 2 using Daphnia magna as test organism. Test concentrations from 0.5 to 2 mg/L were tested using Marlowet R40 as vehicle (2 mg/L). The stability of the highest test concentration (2 mg/L) was analytically examined. A geometric mean value of 0.77 mg/L was measured. No significant effects on reproduction and survival were observed up to the highest concentration tested. Thus, the 21d-NOEC value is determined to be > 0.77 mg/L (measured concentration) and > the solubility in water, respectively.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
13 Jul - 3 Aug 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 202, part 2 adopted in 1982
Deviations:
yes
Remarks:
4 replicates each with 10 daphnids for the solvent control and only two replicates for each test concentration and control
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was mixed with acetone. The test substance was added to the test system as a 10000 fold concentrate in acetone to give a constant level of acetone in each treatment and control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 mg/L v/v
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not stated
Test organisms (species):
Daphnia magna
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Nominal and measured concentrations:
Nominal: 3.2, 10, 32 and 100 µg/L
Measured: > 80% of nominal (80.9 - 90.6%)
Details on test conditions:
TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): Daphnia were transferred into freshly prepared test solutions every monday, wednesday and friday throughout the test.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 4

EFFECT PARAMETERS MEASURED: Number of offspring were counted on day 7, 9, 12, 14, 16, 19 and 21. Number of surviving parental daphnids were recorded at the end of the test.

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: reproduction and survival
Details on results:
No significant effects were observed in all treatments compared to the control.
Validity criteria fulfilled:
not specified
Conclusions:
DEHA had no significant detrimental effect on survival and reproduction of Daphnia magna in the range of water solubility.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study
Qualifier:
according to guideline
Guideline:
other: Draft No.3, ASTM E-47.01 American Society for Testing and Materials, Philadelphia, PA.
Principles of method if other than guideline:
Proposed standard practice for conducting Daphnia magna chronic toxicity tests in a flow-through system.
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Water samples (10 mL) were taken from each test chamber to determine [14C] DOA concentration at days 0, 4, 7, 14 and 21.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: not specified
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.2 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not stated
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Age of parental stock (mean and range, SD): < 24h
- Feeding during test
- Food type/Amount/Frequency: 15 to 30 mL of a S. capricornutum suspension were fed three times daily and 2 mL of trout chow suspension once daily.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
250 +- 25 mg CaCO3/L
Test temperature:
20 °C
pH:
8.1 - 8.3
Dissolved oxygen:
6.2 - 8.6 mg/L
Nominal and measured concentrations:
Measured: 0.014 (+- 0.0003), 0.024 (+- 0.006), 0.052 (+- 0.006), 0.087 (+- 0.02) and 0.18 (+- 0.02) mg/L (= 92.1% of nominal)
Details on test conditions:
TEST SYSTEM
- Type of flow-through: A proportional diluter delivered water and chemical at a rate of four replacement volumes per day for five exposure levels and a solvent control.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Alkalinity: 350 mg/L
- Conductivity: 700 µ 0hm/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: not specified

EFFECT PARAMETERS MEASURED: Survival, reproductive output and growth was recorded after day 21.The surviving adults were removed from the test chamber, isolated on microscope depression slides and measured using a binocular dissecting scope and calibrated eyepiece

VEHICLE CONTROL PERFORMED: yes
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
other: MATC maximum acceptable toxicant concentration
Effect conc.:
0.024 - 0.052 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: mean length, survival and reproduction
Details on results:
- Significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L.
- MATC (maximum acceptable toxicant concentration) for chronic toxicity on Daphnia magna was derived to range between 0.024 and 0.052 mg/L based on statistical analyses of adult meand length, survival and young per adult per reproduction day.
- The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate.
Validity criteria fulfilled:
no
Conclusions:
The results of the 21d-chronic study indicate that the read-across substance bis(2-ethylhexyl) adipate is moderately toxic to Daphnia magna. The mean young per adult per reproduction day after 21 d was significantly afffected in the mean measured exposure levels of 0.087 and 0.18 mg/L. The maximum acceptable toxicant concentration (MATC) range is 0.024 to 0.052 mg/L., based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study with analytical monitoring
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples from the D. magna test were collected on test days 0, 2, 4, 7, 14 and 20 and analyzed for the DEHA. Samples were collected in 500-mL glass bottles with ground glass stoppers (the bottles and stoppers were rinsed three times with small amount of test or acetone control solution which was discarded previously). Samples of diluent water were collected on test days 1 and 8 and samples of solvent control solution were collected on test days 0 and 14 and also analyzed for DEHA. On test days 1, 14 and 21 samples of the test solutions were collected directly from the test chambers after having been in the the test chambers for 24 h (approx. 40 mL from each chamber and combined to provide 400 mL).
The measured initial concentration of DEHA in the test solution at the time of renewal varied from 1.65 to 8.32 µg/L. The majority of the measured values were within 3-6 µg/L. The DEHA concentration in duplicate samples on days 0, 7 and 20 suggest that the concentration of DEHA was homogeneous in the test solutions. The average initial DEHA concentration in all renewal solutions in this limit test was 5 ± 1µg/L.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 1 mL of a DEHA stock solution in acetone (50 mg/L) was added to 3 L of diluent water in a 4-L Erlenmeyer flask (nominal DEHA concentration = 16 µg/L, acetone concentration = 0.3 mL/L). A stir bar was added, the flask covered with sterilized foil, and the mixture vigorously stirred with a vortex height of 1/2 to 3/4 of total solution depth for 24 ± 2 h in the dark at 20 ± 2°C. The DEHA-saturated solution was removed by siphoning through a glass tube that extended from the bottom of the flask through the neck opening. The first several tube volumes of solution were siphoned off and discarded. A solvent control solution, acetone-diluent water, was prepared in the same manner as the DEHA-diluent water test solution.
Test chemical, solvent control and diluent water control solutions were prepared daily.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.3 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not stated
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna neonates, < 4-h old, were obtained from ABS (Aquatic BioSystems, Inc., Fort Collins, CO, USA) and used immediately. At ABS, adult organisms were cultured in moderately hard water (hardness of 102 mg/L (as CaCO3), alkanity of 65 mg/L (as CaCO3), and pH 7.9) at 22°C under a 16/8 (light/dark) cycle and fed a 2:3 mixture of yeast, trout chows and cerophyll (YTC) and green algae (Pseudokirchneriella subcapitata).
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Hardness:
160 - 180 mg/L as CaCO3
Test temperature:
20 ± 1°C
pH:
7.8 - 8.6
Dissolved oxygen:
5.4 - 7.7 mg/L
Nominal and measured concentrations:
Nominal: 16 µg/L
Measured: 5 ± 1 µg/L (initial)
Details on test conditions:
TEST SYSTEM
The test was initiated by placing one young (<4 h) neonate Daphnia magna in each 100 mL glass beaker containing 80 mL of solution. Ten replicates each of diluent water control, solvent control and DEHA solution were used in teh test. Shortly after addition of the daphids, 0.4 mL of a 2:3 undigested YTC:algae mix was added to each chamber. The diluent water control, solvent control and DEHA test solution were renewed daily.

TEST MEDIUM / WATER PARAMETERS
The test diluent waster was city tap water that had been filtered through charcoal and a Millipore system (Milli-Q: Millipore, Billerica, MA, USA), then amended to a hardness of 160-180 mg/L (as CaCO3) with selected salts (USEPA 1994). After the addition and dissolution of the salts, the diluent water was sterilized by filtration (Gelman® pleated capsule with 0.2 µm Versapore® membrane)

OTHER TEST CONDITIONS
The test was performed in a temperature-controlled environmental chamber at 20±1°C. Lightning was controlled to provide a 16/8 light/dark cycle, with a mean illumination of 75±25 ft-c /(807±269 lx)

EFFECT PARAMETERS MEASURED
Each day of the test survival and health (e.g., mobility, presence of ephippia) of the initial organisms were determined. Then, the test organisms were transferred into new test chambers. Neonates were counted each day after the first brood was observed (e.g., on day 7), then discarced. The test was terminated after 21 days of exposure and the cumulative number of neonates determined. The adult females were transferred to individual pre-tarred pans, dried overnight at 74°C and their dry weights measured.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
not performed, limit test
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 4.4 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: reproduction, growth, mortality
Remarks on result:
other: saturated test solution tested
Details on results:
Survival of Daphnia magna was highest in the treatment (100%) compared to both the control (90%) and solvent control (90%). Neonates were first observed in the control and solvent control on day 7, and in the DEHA treatment on day 8. By day 8, 50% of the females in both the control and solvent control and 60% of the females in the DEHA treatment had produced first broods. The average number of neonates per surviving female was 137 for the control and 148 for the solvent control. By comparison, each female exposed to the DEHA solution produced an average of 152 neonates (10% more). Consistent with survival and productivity, the mean dry weight per surviving female was highest in the DEHA exposure (0.804 mg) compared to 0.779 and 0.742 mg for the control and solvent control, respectively.
Three times during the course of the test, the DEHA solution after being in the test chambers for 24 h was collected for analysis. The data for all three samples showed that the concentration of DEHA had decreased during 24 h in the test chambers in the presence of daphnids and food (algae).
Validity criteria fulfilled:
yes
Remarks:
Survival and reproduction of the control organisms surpassed the minimum OECD (1998) and ASTM (2001) criteria for acceptability.
Conclusions:
DEHA had no observed detrimental effect on survival, growth or reproduction in Daphnia magna in the range of water solubility.

Description of key information

Bis(2-ethylhexyl) adipate does not show long-term toxicity to aquatic invertebrates in the range of its water solubility.

Key value for chemical safety assessment

Additional information

Four studies investigating the long-term toxicity of Di(2-ethylhexyl) adipate (CAS 103-23-1, DEHA) to aquatic invertebrates are available. The key study was performed according to GLP and OECD guideline 202, part 2 using Daphnia magna as test organism (Huels 1996). Test concentrations from 0.5 to 2 mg/L were tested using Marlowet R40 as vehicle (2 mg/L). The stability of the highest test concentration (2 mg/L) was analytically examined. A geometric mean value of 0.77 mg/L was measured, which considerably exceeded the intrinsic water solubility of the substance (approx. 0.0032 mg/L, see chapter 4.8). Nevertheless, no significant adverse effects on reproduction rate or survival of the parent animals were observed at any of the concentrations tested. Thus, the 21d-NOEC value is determined to be ≥ 0.77 mg/L (measured concentration) and > the solubility in water, respectively.

 

This result is confirmed by supporting studies: Robillard et al. (2008) conducted a long-term toxicity test with Daphnia magna at the saturation limit, which was measured to be an average exposure concentration of 4.4 μg/L (water solubility = 5.5 µg/Lmeasured using the slow stir method). The test concentration was chosen to avoid insoluble test material and physical entrapment. One hundred percent of the DEHA-treated organisms survived compared to 90% survival in both the control and solvent control. Mean neonate reproduction was 152, 137, and 148 and mean dry weight per surviving female was 0.804, 0.779, and 0.742 mg in the treatment, control and solvent control, respectively. No adverse effects were observed. Thus, the 21d-NOEC value is determined to be ≥ 4.4 µg/L (measured concentration) and > the solubility in water, respectively.

Another supporting study investigated the long-term toxicity of DEHA to Daphnia magna according to GLP and OECD guideline 202, part 2 (Brown 1984). Nominal concentrations of 3.2, 32 and 100 µg/L prepared using acetone (100 mg/L) were tested. The recovery of the test substance analytics was > 80% of nominal. Thus, the effect value was related to the nominal concentration. No significant effects were observed in any treatment compared to the control. Hence, the 21d-NOEC value is determined to be ≥ 100 µg/L and > the solubility in water, respectively.

 

Felder et al. (1986) generated conflicting results. They performed a long-term toxicity test according to ASTM guideline E-47.01 but not under GLP conditions. Test substance concentrations between 0.014 and 0.18 mg/L were prepared using acetone (0.2 mL/L) as vehicle. The authors reported a significantly reduced yield of young per adult per day at mean measured exposure levels of 0.087 and 0.18 mg/L. A MATC (maximum acceptable toxicant concentration) for chronic toxicity on Daphnia magna was determined to be between 0.024 and 0.052 mg/L based on statistical analyses of adult mean length, survival and young per adult per reproduction day. The geometric mean of the LOEC and NOEC was 0.035 mg/L, which is approximately ten-fold above the solubility limit of bis(2-ethylhexyl) adipate. The water solubility measured in this publication (0.78 mg/L) was much higher compared to any of the other studies. Under such conditions the effects observed could have been caused by physical entrapment rather than chemical interactions. Information provided in the results section of the publication is rather scarce, therefore not allowing for an assessment of the extent of impairment daphnids had to face at a given concentration. In addition, no information on dose-response is included. Based on this remaining uncertainty regarding the quality of this result and since there are several reliable studies available giving a unified picture, this result is not considered relevant for the hazard assessment of DEHA.

 

In conclusion all relevant and sufficiently reliable data indicate that DEHA does not adversely affect aquatic invertebrates at long-term exposure in the range of its water solubility.