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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
30 May - 01 June 1980
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study. However, the study was disregarded since globules of the test substance remained insoluble and floated on the surface of the water in all treatments. Daphnia was observed to be trapped in the globules and became immobilized. Thus, adverse effects observed are considered to be caused by physical interaction rather than systemic toxicity. Therefore, the study is rated as invalid for the hazard assessment of DEHA.
Qualifier:
according to guideline
Guideline:
other: US-EPA 660/3-75009: methods of acute toxicity tests with fish, macroinvertebrates and amphibians (1975)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by adding 0.2 g test substance to 10 mL acetone (= 20 mg/mL). The stock solutions were diluted using well water to the respective test concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 0.32 - 100 µL
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Globules of the test substance remained insoluble and floated on the surface of the water in all treatments.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house culture
- Age at study initiation (mean and range, SD): < 24h
- Feeding during test: none

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions (same as test or not): same
- Type and amount of food: suspension of trout chow and alfalfa
- Feeding frequency: daily until 24h before testing
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
255 mg/L as CaCO3
Test temperature:
22 °C
pH:
7.8 - 8.1
Dissolved oxygen:
9.3 - 9.5 mg/L
Nominal and measured concentrations:
Nominal: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass beakers containing 200 mL test solution
- 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): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water
- Alkalinity: 368 mg/L
- Conductivity: 50 µmhos/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature, dissolved oxygen and pH were measured at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:16h light: 8h dark

EFFECT PARAMETERS MEASURED: Immobilisation of daphnids were recorded after 24 and 48h test duration.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: beginning at 0.1 mg/L and increasing the amount of test material by a factor of 10 until a toxic level was found
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.66 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Daphnia was observed to be trapped in the undissolved test material and became immobilized in all treatments.
Details on results:
- Mortality of control: none
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Globules of the test substance remained insoluble and floated on the surface of the water in all treatments.
- Effect concentrations exceeding solubility of substance in test medium: Daphnia was observed to be trapped in the globules and became immobilized.

Table 1: Immobilisation and observations of the different treatments after 24 and 48h exposure time

Nominal concentration [mg/L]

Immobilisation

24h

48h

immobilized

remarks

immobilized

remarks

Control a

Control b

0

0

none

none

0

0

none

none

Solvent control a

Solvent control b

0

0

none

none

0

0

none

none

0.32 a

0.32 b

0

0

clumping*

clumping*

0

0

clumping*

clumping*

1.0 a

1.0 b

0

0

clumping*

clumping*

9

10

clumping*

clumping*

3.2   a

3.2   b

0

0

clumping*

clumping*

10

9

clumping*

clumping*

10.0 a

10.0 b

0

0

clumping*

clumping*

10

10

clumping*

clumping*

32.0 a

32.0 b

0

0

clumping*

clumping*

9

10

clumping*

clumping*

100  a

100  b

1

2

clumping*

clumping*

10

10

clumping*

clumping*

Validity criteria fulfilled:
no
Remarks:
Undissolved test material was not removed from the test system. Based on observations the adverse effects recorded are caused by physical interaction of the daphnids with the undissolved test material rather than systemic toxicity.
Conclusions:
The study is disregarded since globules of the test substance remained insoluble and floated on the surface of the water in all treatments. Daphnia was observed to be trapped in the globules and became immobilized. Thus, adverse effects observed are considered to be caused by physical interaction rather than systemic toxicity. Therefore, the study is rated as invalid for the hazard assessment of DEHA.
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 - 27 Apr 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution was prepared by mixing of 500 mg DEHA/L and 100 mg Tween 80/L. The stock solution was stirred for 10 min at 20 °C. The stock solution was diluted with tap water and 100 mg/L Tween 80 to prepare the different test concentrations. 10 mL of the respective test solution was added to the test vessel.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tween 80
- Concentration of vehicle in test medium (stock solution and final test solution): 100 mg/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISMS
- Source: inhouse culture originally obtained by Institut National de Recherche Chimique Appliquee, France
- Method of breeding: photoperiod: 16 h light / 8 h dark , daily feeding with green algae, once (after the change of water) feeding with yeast
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.44 mmol/L
Test temperature:
18 - 20 °C
pH:
7.41 - 7.84
Dissolved oxygen:
6.47 - 9.10 mg/L
Nominal and measured concentrations:
Nominal: 15.62, 31.2, 62.5, 125, 250, 500 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Centrifuge tube with 10 mL test solution
- No. of organisms per vessel: 5
- 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
- Source/preparation of dilution water: filtered (ac) and dechlorinated tap water
- Ca/mg ratio: 4:1
- Conductivity: 570 µS/cm
- Intervals of water quality measurement: at the beginning and the end of the test

OTHER TEST CONDITIONS
- Photoperiod: 16:8 light: dark
- Light intensity: ~ 5 µE/m*m*s

EFFECT PARAMETERS MEASURED : Immobility was recorded after 3, 6, 24 and 48h exposure time.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
250 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
RS-Freetext:
RESULT IMMOBILISATION
EC 50 (3h): > 500 mg/l
EC 50 (6h): > 500 mg/l
EC 50 (24h): > 500 mg/l
EC 50 (48h): > 500 mg/l

immobilised organisms after 48 hours (from 20 organisms / conc.):
nom. conc. (mg/l ) 0h 3h 6h 24h 48h
(control) - - - - -
(cont. solubilizer) - - - - -
15.62 - - - - 1
31.2 - - - - 1
62.5 - - - - 1
125 - - - - 2
250 - - - - 2
500 - - - - 5

RESULTS SYMPTOMS
At concentrations between 100 and 500 mg/l the test organisms swam partly at the surface.

REMARK
Effect concentration is greater than the solubility of the substance in the test medium.

Behavior of the test item during the test: No remarkable observations.
Validity criteria fulfilled:
yes
Conclusions:
A 48h acute toxicity test according to EU method C.2 using Daphnia magna as test organism revealed that DEHA is not acutely harmful to freshwater invertebrates up to the saturation limit in water.
Executive summary:

The study was performed according to EU method C.2 using Daphnia magna as test organism. A stock solution of 500 mg/L was prepared by mixing with 100 mg Tween 80/L. The stock solution was diluted with tap water and 100 mg/L Tween 80 to prepare the different test concentrations. Nominal test concentrations between 15.62 and 500 mg/L were tested. Immobile daphnids were observed after 48h exposure time. A 48h-EC50 is determined to be > 500 mg/L. At concentrations between 100 and 500 mg/L the test organisms swam partly at the surface of the test vessels. Since the effect concentrations are greater than the solubility of DEHA in the test medium, it can be concluded that DEHA is not harmful to freshwater invertebrates up to the saturation limit in water.


Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20 - 26 Nov 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Undissolved test material was not removed from the test system: however, retention chambers were used to prevent direct contact of the test organisms to the undissolved test material (Mysidopsis bahia, Paleomonetes pugio).
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 797.1930 (Mysid Acute Toxicity Test)
Principles of method if other than guideline:
Acute toxicity test with three marine invertebrates. the protocol closely follows the EPA/OTS guidelines as described in "Mysid shrimp acute toxicity test". Federal register.1985. Mysid shrimp acute toxicity test. Vol. 50, No. 188, Sep 27, 1985, pp. 39367-39369
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Ths control and the high, mlddle and low test concentratlons (A and B replicates) were sampled and analyzed for DOA concentration prior to the start of the definitive exposure. Results of these pretest analyses were used to judge whether sufficient quantities of test material were being delivered and maintained in the exposure aquaria to initiate the definitive test. During the test, water samples were removed from both replicate test solutions of each treatment level and the controls on days 0 and 4 for analyses of DOA.
Each exposure solution sample was collected from the approximate midpoint of the aquarium using a siphon comprised of glass ans Silastic tubing. In addition, three quality assurance blind samples were prepared at each sampling interval and remained with the set of exposure solution samples through analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A clear, colorless stock solution of 10 mg/mL was prepared by diluting 2.5011 grams (g) of DOA with acetone to volume in a 250 mL volumetric flask. A Sage Syrlnge Pump (Model #355) was used to deliver the stock solution into the diluter system where it was diluted to provide the desired exposure concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 100 µL/L (acetone concentration in the highest treatment level)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Throughout the exposure period, the two highest treatment levels (1.0 and 0.65 mg/L, nominal) contained a clear, colorless oily fim on the surface of the test solutions. Treatments below these levels, 0.42, 0.27 and 0.18 mg/L, were clear and colorless and obtained no visible surface film or other sign of insoluble test material.
Test organisms (species):
other aquatic crustacea: Mysidopsis bahia, Paleomonetes pugio, Ampelisca abdita
Details on test organisms:
TEST ORGANISM
- Name: Mysidopsis bahia
- Source: in-house culture
- Age at study initiation (mean and range, SD): < 24h
- Feeding during test: brine shrimp nauplii daily

ACCLIMATION
- Acclimation period: none (in-house culture)
- Type, amount and feeding frequency of food: brine shrimp nauplii daily and hatchfry Encapsulon three times weekly

- Name: Paleomonetes pugio
- Source: obtained from SP lnc. a commercial supplier in Massachusetts, US
- Age at study initiation (mean and range, SD): < 24h
- Length at study initiation (length definition, mean, range and SD): 22 mm (17 - 28 mm)
- Feeding during test: none

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same
- Type, amount and feeding frequency of food: dry commercial pelleted food, ad libitum once daily

- Name: Ampelisca abdita
- Source: field collected from Bourne Cove, Wareham, Massachusetts on 10 November 1988.
- Age at study initiation (mean and range, SD): < 24h
- Length at study initiation (length definition, mean, range and SD): 5.4 mm (3.8 - 7.6 mm)
- Feeding during test: none

ACCLIMATION
- Acclimation period: 6 days
- Acclimation conditions (same as test or not): same
- Type, amount and feeding frequency of food: Each vessel was fed 200 mL of a marine algae (isochrysis galbana or Tetraselmis maculata) on each weak day.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21 - 22 °C
pH:
7.5 - 8.1
Dissolved oxygen:
4.9 - 8.1 mg/L
Salinity:
31 - 35 ‰
Nominal and measured concentrations:
Nominal: 0.18, 0.27, 0.42, 0.65 and 1.0 mg/L
Measured (arith. mean): 0.049, 0.053, 0.053, 0.083 and 0.23 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type of flow-through: proportional diluter
- Renewal rate of test solution (flow rate): 13 aquarium volumes per day
- 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): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was prepared by fllterlng natural seawater collected from the Cape Cod Canal, Bourne, Massachusetts, US. In the laboratory, the seawater was passed through a series of polypropylene core filters (20- and 5-mlcron) end then reclrculated withln an epoxy-lined concrete reservoir prior to use.
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dlssolved oxyygen concentratlon, pH, salinity and temperature were measured once daily in each replicate of the controls and each treatment level.

OTHER TEST CONDITIONS
- Photoperiod: 16h light: 8h dark
- Light intensity: 20 - 70 footcandles

EFFECT PARAMETERS MEASURED: Mortality was recorded after 24, 48, 72 and 96h test duration.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: no mortality of Mysidopsis bahia was observed up to the highest concentration tested
Details on results:
- Biological observations: During the 96-hour definitive test, no significant toxicant related mortalities occurred among the three species exposed to DOA concentrations of <= 0.23 mg/L. Following 96- hours of exposure to the highest treatment level (0.23 mg/L) mortality of 0 and 5% was
observed among the exposed mysid shrimp and grass shrimp, respectively. Mortality ranged from 10 - 50% among Ampelisca exposed to all treatment levels and the controls. The mortalites observed among the exposed Ampelisca occurred within the initial 24 hours of the test and did not establish a concentration-effect relationship. During the initial 24h of the study many of the exposed Ampelisca, at all treatment levels and controls,
become entrapped in the surface of the test solutions. No organisms were observed on the solution surface after the intial 24h. Since Ampelisca are benthic organisms which are generally found burrowed in sediment, the sporadic mortalities observed during this study are considered to be a response (entrapment) to the exposure conditions (i.e., absence of sediment) and not related to system toxicity of the test substance.




No mortality was recorded in any treatment and control throughout the the test duration of 96h.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Throughout the exposure period a clear colorless oily film was observed on the surface of the two highest treatment levels (0.083 and 0.23 mg/L).

Tab1: Mortality of marine crustacea and observations after 24 and 96h test duration

Mean measured concentration [mg/L]

Cumulative mortality [%]

24h

96h

A

B

mean

A

B

mean

Mysidopsis bahia

Control 1

Control 2

0

0

0

0

0

0

0

0

0

0

0

0

Solvent control

0

0

0

0

0

0

0.049

0

0

0

0

0

0

0.053

0

0

0

0

0

0

0.053

0

0

0

0

0

0

0.083*

0

0

0

0

0

0

0.23*

0

0

0

0

0

0

Paleomonetes pugio

Control

0

0

0

10

10

10

Solvent control

0

0

0

0

0

0

0.049

0

0

0

0

0

0

0.053

0

0

0

0

10

5

0.053

0

0

0

0

0

0

0.083*

0

0

0

10

10

10

0.23*

0

0

0

0

10

5

Ampelisca abdita

Control

30

10

20**

30

10

20

Solvent control

30

30

30**

30

30

30

0.049

0

20

10**

0

20

10

0.053

30

10

20**

30

10

20

0.053

10

10

10**

10

10

10

0.083*

30

20

25**

30

20

25

0.23*

30

20

25**

30

20

25

*: Throughout the 96h exposure a clear, colorless oily film existed on the solution surface.

**: During the initial 24h of the exposure period, several of the surviving organisms were on the surface of the test solution.

Validity criteria fulfilled:
yes

Description of key information

No toxicity is observed within the limit of water solubility.

Key value for chemical safety assessment

Additional information

Two studies investigating the short-term toxicity of Bis(2-ethylhexyl) adipate (CAS 103-23-1, DEHA) to freshwater invertebrates and one study to marine invertebrates are available.

The key study was performed according to EU method C.2 using Daphnia magna as test organism (BASF 1988). A stock solution of 500 mg/L was prepared by mixing with 100 mg Tween 80/L. The stock solution was diluted with tap water and 100 mg/L Tween 80 to prepare the different test concentrations. Nominal test concentrations between 15.62 and 500 mg/L were tested. Immobile daphnids were observed after 48h exposure time. A 48h-EC50 is determined to be > 500 mg/L. At concentrations between 100 and 500 mg/L the test organisms swam partly at the surface of the test vessels. Since the effect concentrations are greater than the solubility of DEHA in the test medium, it can be concluded that DEHA is not acutely harmful to freshwater invertebrates up to the saturation limit in water.

This conclusion is confirmed by a test investigating the short-term toxicity of DEHA to the marine invertebrates Mysidopsis bahia and Paleomonetes pugio. The study was performed similar to EPA/OTS guideline 797.1930 (Mysid Acute Toxicity Test) and GLP using three marine crustacean species (Mysidopsis bahia, Paleomonetes pugio and Ampelisca abdita) as test organisms (Suprenant 1989). Test concentrations between 0.18 and 1.0 mg/L were prepared using acetone as vehicle. Test substance analytics revealed slightly lower concentrations of 0.049 to 0.23 mg/L. During the 96-hour definitive test, no significant toxicant related mortalities occurred among the two species Mysidopsis bahia and Paleomonetes pugio. Following 96h of expose to the highest treatment level (0.23 mg/L) mortality of 0 and 5% was observed among the exposed mysid shrimps and grass shrimps, respectively. In contrast, mortality ranged from 10 - 50% among Ampelisca exposed to all treatment levels and the controls. The mortalities observed among the exposed Ampelisca occurred within the initial 24h of the test and did not establish a concentration-effect relationship. During the initial 24h of the study many of the exposed Ampelisca, at all treatment levels and controls, became entrapped in the surface of the test solutions. No organisms were observed on the solution surface after the initial 24h. Since Ampelisca is a benthic organism, which is generally found burrowed in sediment, the sporadic mortalities during this study are considered to be a response to the exposure conditions (interaction with undissolved test material, absence of sediment) and not related to the systemic toxicity of DEHA. Thus, the results for Ampelisca abdita are rated to be invalid for the hazard assessment of DEHA.

A third study was performed according to US-EPA guideline 660/3-75009: methods of acute toxicity tests with fish, macroinvertebrates and amphibians (1975) under GLP conditions (Forbis and Boudreau 1980). Test concentrations between 0.32 and 100 mg/L were prepared using acetone as vehicle. The test concentrations were not verified analytically. 9 to 10 (of 10) daphnids in all treatments except for the 0.32 mg/L treatment were dead after an exposure time of 48h. The 96h-EC50 value for immobilisation of Daphnia magna is determined to be 0.66 mg/L. However, globules of the test substance remained insoluble and floated on the surface of the water in all treatments. Daphnia was observed to be trapped in the globules and became immobilized. Thus, adverse effects observed are considered to be caused by physical interaction of the daphnids with the test substance rather than systemic toxicity. Therefore, the study is rated as invalid for the hazard assessment of DEHA.