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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:
Apr 1976 - Feb 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
no
Remarks:
study pre-dates GLPs
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations on day 0, and every 3-5 days throughout the 21-day test.
- Sampling method: details not available in study report
- Sample storage conditions before analysis: Preserved with 1% formaldehyde, and stored at 5 C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct dilution of the appropriate amount of test substance into blended well water
- Controls: Blended well water
- Evidence of undissolved material: No
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: N/A
- Source: In-house culture
- Age of parental stock: <12 h
- Feeding during test: yes
- Food type: ground trout chow/alfalfa mixture, 1000 mg/L
- Amount: 5 mg/L
- Frequency: Each diluter cycle

ACCLIMATION
- Acclimation period: Not specified
- Acclimation conditions (same as test or not): N/A
- Type and amount of food: N/A
- Feeding frequency: N/A
- Health during acclimation (any mortality observed): N/A

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Young were manually siphoned from existing culture units.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
120 +/- 18 mg/L as CaCO3
Test temperature:
21 +/- 1 C
pH:
7.5 +/- 0.2
Dissolved oxygen:
8.9 +/- 0.4 mg/L
Nominal and measured concentrations:
The nominal test concentrations are not specified in the study report. Mean measured concentrations were 0, 0.13, 0.27, 0.47, 0.93, and 1.94 mg a.i./L.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material: 1 liter glass beakers, 1 L test solution
- Aeration: N/A (test was flow-thru)
- Type of flow-through: Mount and Brungs proportional diluter, with modifications
- Renewal rate of test solution: 125 ml per 20 min cycle, or one chamber volume every 2.5 hr.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 5 daphnids/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Blended well water (a mixture of well water and deionized water)
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Salinity: N/A
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: N/A
- Light intensity: N/A

EFFECT PARAMETERS MEASURED: Mortality, 24 hr, 96 hr, 7 days, then daily to end of test. Reproduction, daily beginning day 7 or 8 to end of test.

RANGE-FINDING STUDY: no
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Parent (Fo) Survival and Reproduction (Mean Brood Size; Total # of young)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Total young production
Remarks on result:
other: 95% CL 0.22-0.54 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Average brood size
Remarks on result:
other: 95% CL 0.23-0.60 mg/L
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Percentage of days reproduction occurred
Remarks on result:
other: 95% CL 0.31-0.76 mg/L
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 0.56-0.94 mg/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 0.82-1.66 mg/L
Details on results:
- Mortality of parent animals: Controls, 10%
- No. of offspring produced per day per female: N/A
- Body length and weight of parent animals: N/A
- Type and number of morphological abnormalities: N/A
- Type and number of behavioural abnormalities: N/A
- Number of males and females (parental): N/A
- Time to first brood release or time to hatch: 7 or 8 days
- Brood size: Controls, 8.05 (mean).
- Time to sexual maturity: N/A
- Type and magnitude of biochemical changes: N/A
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
All results were calculated with a computerized probit analysis program after Finney (1964, 1971). Survival data for 96 hr and 21 days was used to calculate LC50 and EC50 values and confidence limits. All results were calculated based on mean measured concentrations of test material.

Chronic Toxicity of Alkyl Ethoxy Sulfate (AES)* to Daphnia magna (study # 20710)

Test Concentration (mg/L)

Mortality (%)

Total Young Produced

Mean Brood Size

% of Days that Reproduction Occurred

0

10

783

8.0

92

0.13

45

No data **

No data **

No data **

0.27

35

529

6.2

100

0.47

30

334

3.5

75

0.93

50

0

0

0

1.94

100

0

0

0

* C14-16EO2.25S.

Results are cumulative at 21 days.

Test concentration is mean measured concentration.

Statistical significance not specified in study report.

** Malfunction in feeding system caused no feed to be delivered to this test concentration.

Validity criteria fulfilled:
yes
Conclusions:
The 21 day NOEC of C14-16EO2.25S (alkylethoxysulfate, AES) to Daphnia magna was 0.27 mg/L, based on measured concentrations. The NOEC was based on reproduction. 
Executive summary:

A 21 day chronic toxicity test of C14 -16EO2.25S (alkylethoxysulfate, AES) was conducted using Daphnia magna in a flow-through test. Mean measured test concentrations were 0, 0.13, 0.27, 0.47, 0.93, and 1.94 mg a.i./L. Four replicates per concentration, 5 organisms per replicate, were tested, using a modified Mount and Brungs proportional diluter. Survival was monitored at 24 hr., 96 hr., 7 days, and daily thereafter. Reproduction was monitored beginning with the production of the first brood on day 7 or 8, and daily thereafter.

The 21 -day NOEC, 0.27 mg/L, was based on both survival and reproduction.

EC50's for reproduction, and an LC50 for mortality, were also calculated and are available in the study report.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the Category Approach Justification provided in Section 13
Reason / purpose for cross-reference:
read-across source
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Parent (Fo) Survival and Reproduction (Mean Brood Size; Total # of young)
Remarks on result:
other:
Remarks:
Source, key, 68585-34-2, 1977a
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Total young production
Remarks on result:
other: 95% CL 0.22-0.54 mg/L
Remarks:
Source, key, 68585-34-2, 1977a
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Average brood size
Remarks on result:
other: 95% CL 0.23-0.60 mg/L
Remarks:
Source, key, 68585-34-2, 1977a
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Percentage of days reproduction occurred
Remarks on result:
other: 95% CL 0.31-0.76 mg/L
Remarks:
Source, key, 68585-34-2, 1977a
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.74 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 0.56-0.94 mg/L
Remarks:
Source, key, 68585-34-2, 1977a
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.17 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL 0.82-1.66 mg/L
Remarks:
Source, key, 68585-34-2, 1977a
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 Sep - 07 Oct 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Study predates guideline
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were removed from two replicate vessels of the high, medium, and low test concentrations on two days prior to initiating the chronic exposure. During the in-life phase of the test, water samples were removed and analyzed for the test substance weekly from two replicate vessels of each test concentration and the control.
- Sampling method: For the in-life phase of the test, water samples were removed and analyzed for the test substance weekly from two replicate vessels of each test concentration and the controls. Two quality assurance (QA) samples, formulated in test dilution water to a concentration unknown to the analyst, were also prepared at each sampling interval. The QA samples remained with the samples of the exposure solutions through extraction and analysis. Analyses of the QA samples were used to determine the accuracy and the quality control maintained during analytical procedures. Samples were analyzed for the test substance spectrophotometrically using a Standard Methods procedure validated by SBI prior to test initiation. A method validation and recovery study established an average recovery of 103 +- 9 %, as Methylene Blue Active Substance (MBAS), in the test concentration range of interest.
- Sample storage conditions before analysis: N/A
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Culture and test dilution water were prepared by fortifying well water according to the formula for hard water (ASTM, 1980), and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. This water generally had a total hardness and alkalinity as calcium carbonate (CaCO3) of 160-180 mg/L and 110-130 mg/L, respectively, a pH range of 7.9 - 8.3, a temperature of 20 +- 1 degree C, a dissolved oxygen concentration of greater than 60% of saturation and a specific conductivity of 400-600 micromhos per centimeter (umhos/cm).
- Controls: fortified well water
- Evidence of undissolved material: Throughout the 21-day exposure period no visible sign of insoluble material (e.g. film of material on the surface of the test solution, precipitate) was observed in any of the test solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: N/A
- Source: Springborn Bionomics Culture Facility, Wareham Massachusetts
- Age of parental stock: Offspring produced during the 24 hour period prior to the start of the 21 day exposure were used to initiate the study.
- Feeding during test
- Food type: suspension of Fleishmann's yeast (5 mg/mL) and a suspension of green algae (Ankistrodesmus falcatus; 4.0 X 10 ^7 cells/mL).
- Amount: Fleishmann's yeast 0.5 mL and Ankistrodesmus falcatus 2.0 mL
- Frequency: The food was introduced three times daily on weekdays and twice daily on weekends and holidays.

ACCLIMATION
- Acclimation period: N/A
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: N/A
- Feeding frequency: N/A
- Health during acclimation (any mortality observed): N/A
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
160-180 mg/L CaCO3
Test temperature:
18-21 degrees C
pH:
7.6 - 8.3
Dissolved oxygen:
8.4 to 9.1 mg/L ( means over the series of concentrations). DO was greater than 60% of saturation throughout the study.
Nominal and measured concentrations:
Nominal test concentrations were 0, 0.19, 0.38, 0.75, 1.5, and 3.0 mg a.i./L.

Mean measured concentrations were: <0.025, 0.16, 0.18, 0.33, 0.92, and 2.4 mg/L.

Measured concentrations were 44-80% of nominal. Results are based on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass battery jars
- Type: open
- Material: glass, test solutions drained from the vessel through a 3.5 X8.0 cm notch on the upper edge of the jars. The drains were covered with a Nitex 40-mesh screen to prevent loss of the daphnids. The vessel volume capacity was -1.8 liters.
- Aeration: test was flow-through
- Type of flow-through: A 200-mL Mount and Brungs proportional diluter
- Renewal rate of test solution: 4.5 aquarium volumes per 24 hour period
- No. of organisms per vessel: 20 (note: there was a total of 80 daphnids/test concentration)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 20 daphnids per ~1.8 L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Culture and test dilution water were prepared by fortifying well water according to the formula for hard water (ASTM, 1980), and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: 110-130 mg/L CaCO3
- Ca/mg ratio: N/A
- Conductivity: 400-600 micromhos per centimeter
- Culture medium different from test medium: No
- Intervals of water quality measurement: The test solution temperature was measured daily in one replicate vessel of each treatment level and the control solution throughout the 21-day exposure. Dissolved oxygen concentration of the test solutions was measured every weekday in one replicate vessel of each treatment level and the control. Total hardness, alkalinity, specific conductivity and pH of the test solutions were monitored weekly in one aquarium from each treatment and the control solution.

OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: 16 hours of light and 8 hours of darkness
- Light intensity: The test area was illuminated with Cool White and Grow Lux fluorescent lights at an intensity of 15 to 75 foot-candles.

EFFECT PARAMETERS MEASURED: Survival and reproduction were measured on day 3 and every weekday from day 7 through day 21.

RANGE-FINDING STUDY: no
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: geometric mean based on 0.18 mg/L (NOEC) and 0.33 mg/L (LOEC)
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: geometric mean based on 0.18 mg/L (NOEC) and 0.33 mg/L (LOEC)
Details on results:
- Mortality of parent animals: No daphnids survived the 21 day exposure to the two highest test concentrations (0.92 and 2.4 mg/L). Survival among daphnids exposed to 0.33 mg/L of test substance was 41+- 19% which were significantly (P=0.05) different from the survival (89 +- 3%) of the control organisms. Exposure to the test substance concentrations <= 0.18 mg/L did not affect the survival of the test organism.
- No. of offspring produced per day per female: No daphnids survived the 21 day exposure to the two highest test concentrations (0.92 and 2.4 mg/L). Reproduction among daphnids exposed to 0.33 mg/L of test substance was 3 +-2 which were significantly (P=0.05) different from the reproduction (71+-4) of the control organisms. Exposure to the test substance concentrations <= 0.18 mg/L did not affect the reproduction of the test organism.
- Body length and weight of parent animals: N/A
- Type and number of morphological abnormalities: N/A
- Type and number of behavioural abnormalities: N/A
- Number of males and females (parental): N/A
- Time to first brood release or time to hatch: N/A
- Egg development time: N/A
- Brood size: N/A
- Time to sexual maturity: N/A
- Type and magnitude of biochemical changes: N/A
- Other biological observations: N/A
- Effect concentrations exceeding solubility of substance in test medium: no
Reported statistics and error estimates:
Weekly survival data, transformed to arc sin percentage and determinations of cumulative production of offspring per female derived during the chronic toxicity test, were subjected to a one way, single classification analysis of variance. If significant (P=0.05) differences between treatments were observed, Dunnett's procedure was used to determine which treatments, if any, were significantly different from the control.

The theoretical threshold concentration expected to produce no deleterious effects was estimated as the Maximum Acceptable Toxicant Concentration (MATC). The MATC was calculated by taking the geometric mean of the limits set by the lowest test concentration that showed a statistically significant effect (Lowest Observed Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant difference from the control (No Observed Effect Concentration, NOEC). Based on these data, the MATC of the test substance to D. magna was estimated.

Chronic Toxicity of AES* to Daphnia magna (study 34679)

Test Concentration (mg/L; measured)

Mortality (%)

Reproduction (offspring/female)

0

11

71

0.16

12

65

0.18

22

61

0.33

59 **

3 **

0.92

100 **

-- **

2.4

100 **

-- **

*AES = alkylethoxysulfate. The specific compound tested was C14-15EO2.25S.

** Significantly different from control (P=0.05).

The chronic Daphnia test was 21 days in duration.

Validity criteria fulfilled:
yes
Conclusions:
The 21-day chronic NOEC of C14-15EO2.25S (alkylethoxysulfate, AES) to Daphnia magna was 0.18 mg/L, based on measured test concentrations. The NOEC is based on both mortality and reproduction.
Executive summary:

In a 21 -day chronic test, Daphnia magna were exposed to C14 -15E2.25S (alkylethoxysulfate, AES) under flow-through conditions. The nominal test concentrations were 0, 0.19, 0.38, 0.75, 1.5, and 3.0 mg a.i./L. Mean measured concentrations were 0, 0.16, 0.18, 0.33, 0.92, and 2.4 mg/L. Measured concentrations were 44 -80% of nominal. Results are based on measured concentrations.

Survival and reproduction were recorded during the exposure to determine the effect of the test substance on Daphnia magna. Daphnid survival (89%) in the control exceeded the standard performance criteria for survival (80%). Daphnid reproduction (71 offspring per female) exceeded the standard performance criteria for reproduction (60 offspring per female).

Based on both mortality and survival, the 21 -day NOEC of C14 -15EO2.25S (alkylethoxysulfate) to Daphnia magna was 0.18 mg/L (measured concentrations). The LOEC was 0.33 mg/L.

The maximum acceptable toxicant concentration (MATC) after 21 days exposure was 0.24 mg/L of the test substance (geometric mean of NOEC and LOEC).

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 Jul 1992 - 01 Feb 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Series on Testing and Assessment, Number 53: Guidance Document on Simulated Freshwater Lentic Field Tests (Outdoor Microcosms and Mesocosms), ENV/JM/MONO(2006)17, Organization for Economic Cooperation and Development, Paris France.
Deviations:
yes
Remarks:
: Adapted for the indoor, flowing stream channels in P&G's Experimental Stream Facility, fed by continuous flow of unfiltered water from an adjacent, high quality stream.
Principles of method if other than guideline:
Study was part of a much larger mescosm study in the P&G Experimental Stream Facility (ESF). This study was performed according to ESD Study Protocol E92-008. Snails (Elimia sp.) were exposed to nominal concentrations of 0, 12, 37, 111, 333 and 1000 µg active AES/L in the tailpools of the experimental streams for 8 weeks. Organisms were observed for mortality, length and weight gain weekly after test initiation. Results were analyzed for the calculation of NOEC.
GLP compliance:
yes
Remarks:
EPA GLP
Analytical monitoring:
yes
Details on sampling:
- Concentrations: weekly from the tail pools of all stream channels
- Sampling method: grab samples
- Sample storage conditions before analysis: Weekly grab samples were preserved with 3% formalin (v/v of 37% formaldehyde) and refrigerated until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test material dosing system required the AES to be prepared as a highly concentrated aqueous solution in a chemical feed tank. This concentrate was then pumped into the river water distribution system that passed through an in-line mixing baffle and entered the experimental stream. The concentrated stock solutions were prepared in deionized water to achieve the desired in-stream nominal concentrations in the experimental streams. A recharging cycle of seven days utilizing chemical feed tanks in a reciprocating manner was used to provide constant delivery of the test material over the course of the eight week study.

The AES concentrates were prepared in 416 L stainless steel chemical feed tanks fitted with variable speed stirrers. The chemical feed tanks were half filled with deionized water, and the stirrers started at a very low speed. The AES was slowly added to minimize dust formations and maximize wetting of powder. The AES was allowed to wet totally and stirred prior to further addition of deionized water. The addition of water was achieved during this process via a computerized flow controller. After the AES was totally wetted, deionized water was added to bring total volume in the chemical feed tank to 416 L. The speed of the stirrers was increased and the AES allowed to mix thoroughly for at least 24 hours prior to use or sampling. The AES was then pumped out of the bottom outlet tube into the river water stream flow via a Pulsa Series Model 340 diaphragm pump.
- Controls: River water pumped continuously from the LEFR.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no
Test organisms (species):
other aquatic mollusc: Elimia sp.
Details on test organisms:
TEST ORGANISM
- Common name: Snail
- Strain/clone: Not reported
- Source: Snails were collected on Aug. 14, 1992 from a riffle (depth of <0.3 m) in the Lower East Fork located between the ESF intake and the discharge of the Clermont County Lower East Fork Wastewater Treatment Plant. Snails were found on flat stones covered with diatom films and mud. Approximately 300 snails (0.6 - 1.2 cm shell length, 0.3 - 0.6 cm opercular opening) were collected and maintained in the Living Stream holding tank of ESF.
- Shell length at study initiation: 10.3-16.6 mm (measured by Vernier calipers)
- Weight at study initiation: 1.212-4.900 g (measured by Mettler AE100 digital electronic analytical balance)
- Feeding during test: Yes
- Food type: Natural, provided from natural river in-flow and colonization on cobbles.
- Amount: Variable, depending on what was available from the river, what colonized the cobbles, and on
invertebrates, community interactions (intra- and inter-specific competition), predation, trophic feeding level, timing of emergence, etc.
- Frequency: Ad libitum, according to what was available in the stream mesocosm.

ACCLIMATION
- Acclimation period: 10 d
- Acclimation conditions: same as test
- Type and amount of food: See above
METHOD FOR PREPARATION AND COLLECTION: Snails were found on flat stones at a riffle (depth of < 0.3 m) in the Lower East Fork River covered with diatom films and mud. Approximately 300 snails (0.6 - 1.2 cm shell length, 0.3 - 0.6 cm opercular opening) were collected and placed in the Living Stream holding tank of ESF. Snails were measured for initial shell length and total body weight.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
8 wk
Hardness:
Not specified in the study report. Hardness was within historical ranges observed at the ESF. Hardness ranged 100-180 mg/L in the 112 day period of testing another anionic surfactant (LAS) (mean hardness 147).
Test temperature:
Approx. 12.5-25.5 °C
pH:
Approx. 7.9-8.3
Dissolved oxygen:
Approx. 6-9.5 mg/L
Nominal and measured concentrations:
Nominal test concentrations were: 0 (control), 12, 37, 111, 333 and 1000 µg active AES/L.
Measured concentrations in the tail pools were: 0, 14±7, 27±7, 75±49, 234±72, and 730±151 µg/L. Measured concentrations were 68-117% of nominal. Results are based on measured concentrations.

Note: measured concentrations at the head of the streams were: 0, 13, 34, 68, 251, and 774 ug/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Tail pool (1.2 m long, 0.6 m deep, 568 L volume) section of stream channel of the Experimental Stream Facility. Four 2 mm(2) wire mesh baskets containing clams were placed into the tail pool section of each stream.
- Type: Open
- Type of flow-through: Experimental Stream Facility (ESF) stream channels with current velocity of approx. 0.5 m/s. A flow controller was attached to each head tank to provide constant, non-impinged flow at the desired level.
- Renewal rate of test solution: 166 L/min per stream
- No. of organisms per vessel: 5 snails/wire mesh basket
- No. of vessels per concentration (replicates): 4 wire mesh baskets per tail pool (20 snails total).
- No. of vessels per control (replicates): 4 wire mesh baskets (2 control streams with minor alterations were used in the study)
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: River water for ESF was obtained from the Lower East Fork, Little Miami River, Clermont County, Ohio. This river is rated as an exceptional warm water fishery by the Ohio EPA (OEPA 1985) and drains into a national and state scenic river. Several endangered molluscs and fish are residents in the drainage.
- Total organic carbon: 4.8±0.9 mg/L
- Dissolved organic carbon: 6.2±3.2 mg/L
- Ammonia-Nitrogen: 0.5±0.4 mg/L
- Total Kjehldahl Nitrogen: 1.5±1.8 mg/L
- Nitrate-Nitrogen: 1.5±1.0 mg/L
- Ortho-Phosphorous: 0.17±0.08 mg/L
- Total-Phosphorous: 0.23±0.08 mg/L
- Sulfate: 45±7 mg/L
- Potassium: 7±6 mg/L
- Magnesium: 12±4 mg/L
- Sodium: 13±5 mg/L
- Conductivity: 175-345 µmhos/cm
- Chloride: 20±5 mg/L
- Alkalinity: 136±60 mg/L
- Culture medium different from test medium: No
- Intervals of water quality measurement: Dissolved oxygen, temperature, pH, and conductivity were continuously monitored (at least once every 5 min). This data was logged into electronic data bases by an on-site mini-computer. Total suspended solids were recorded twice weekly.

OTHER TEST CONDITIONS
- Photoperiod: Natural photoperiod specific to the latitude and longitude were used and were adjusted daily through on-site computer control systems. Lights were staged up and down to simulate dawn and dusk.
- Light intensity: 68 µmol/s/m2 accomplished by thirty 1000-watt metal halide arc lamps.

EFFECT PARAMETERS MEASURED: Snails were removed from cages each week and the shell was blotted dry. Mortality, size (shell length) and growth (weight) were recorded and then they were returned to their respective cages and placed back into the pools. Dead snails identified by parted valves or odor were removed.
Length was recorded to determine which snail had died and as an aid in identifying individuals.
During the study, recording snail length was too variable to use reliably as the lip of the shell near the opercular flap eroded. Thereafter the weight of all the snails in the entire basket was used and the average weight of the basket was used to record the growth of snails.
Reference substance (positive control):
not specified
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
730 µg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
(weight gain)
Details on results:
- Mortality: No appreciable mortality was recorded. It ranged from 0 - 10 % independent of AES concentration.
For details, please refer ‘Table 1’ under 'Any other information on results incl. table' section
- Body length and weight: At 8 weeks, snails increased in weight by 54.7 - 94.9 mg wet weight. Snails exposed to AES did not differ from the control treatment at any time, but significant differences were indicated by analyses of variance beginning on Day 42, presumably due to differences between the group exposed to 75 µg/L (which grew the most) and 730 µg/L (which grew the least).
For details, please refer ‘Table 2’ under 'Any other information on results incl. table' section.
Reported statistics and error estimates:
All endpoints (weight gain, length gain, mortality) were evaluated by parametric analysis of variance (SAS Version 6.08) and non-parametric Kruskal-Wallis. Exposure-response evaluations included linear and quadratic regression analysis. Significance for all statistical tests was inferred at α= 0.05

Table 1: Mortality of snails (Elimia sp.) during an 8-week exposure to Alkylethoxysulfate (AES) (Study # 35522)

Measured Test Concentration (µg AES/L)

Day

7

15

21

28

35

42

49

56

0 (control)

0±0

10±20

10±20

10±20

10±20

10±20

10±20

10±20

VTS (control #2)

10±20

10±20

10±20

10±20

10±20

10±20

10±20

10±20

14±7

10±20

10±20

10±20

10±20

10±20

10±20

10±20

10±20

27±7

5±10

5±10

5±10

5±10

5±10

5±10

10±12

10±12

75±49

0±0

5±10

5±10

5±10

5±10

5±10

5±10

5±10

234±72

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

730±151

0±0

5±10

5±10

5±10

5±10

5±10

5±10

5±10

ANOVA-F

0.67

0.50

0.50

0.50

0.50

0.50

0.41

0.41

p-value

0.677

0.578

0.578

0.578

0.578

0.578

0.867

0.867

Table 2: Weight gain of snails (Elimia sp.) during an 8-week exposure to Alkylethoxysulfate (AES) (Study # 35522)

 Measured Test Concentration (µg AES/L)

Day

7

15

21

28

35

42

49

56

0 ( Stream 3)

2.6 ± 15.7

62.0 ± 63.3

48.7 ± 16.7

57.9 ± 24.5

67.8 ± 20.7

74.7 ± 21.8

79.2 ± 23.9

76.4 ± 27.8

0 ( Stream 6)

8.4 ± 15.0

27.2 ± 14.5

39.2 ± 9.4

51.8 ± 6.1

59.5 ± 5.7

62.5 ± 5.3

67.6 ± 6.5

65.3 ± 4.6

14±7

24.9 ± 12.6

45.3 ± 14.9

49.2 ± 22.0

56.8 ± 20.8

66.2 ± 22.1

69.7 ± 22.6

72.9 ± 22.5

70.0 ± 22.4

27±7

17.2 ± 2.8

28.9 ± 4.8

39.4 ± 4.5

39.5 ± 6.2

51.1 ± 5.3

56.6 ± 6.3

51.6 ± 21.6

46.3 ± 22.9

75±49

27.0 ± 18.1

45.1 ± 22.9

58.0 ± 18.8

74.2 ± 24.5

84.6 ± 22.3

89.6 ± 22.1

95.6 ± 23.6

94.9 ± 21.0

234±72

15.4 ± 4.0

27.9 ± 8.0

40.0 ± 8.1

46.0 ± 10.8

47.8 ± 12.2

54.6 ± 13.5

59.4 ± 12.4

56.9 ± 16.3

730±151

13.7 ± 2.4

33.4 ± 5.1I

45.1 ± 7.3

47.2 ± 7.8

54.2 ± 8.5

57.3 ± 9.2

58.2 ± 8.2

54.7 ± 8.2

ANOVA-F

2.1

0.92

1.00

1.91

2.61

2.46

2.65

2.8

p-value

0.097

0.501

0.45

0.127

0.047

0.059

0.045

0.037

Kruskal-Wallis

16.04

8.56

5.16

10.61

12.06

10.80

13.21

13.14

p-value

0.014

0.201

0.524

0.102

0.062

0.096

0.041

0.042

Validity criteria fulfilled:
not applicable
Conclusions:
The 8 week NOEC of C14-15EO2.25S (Alkylethoxylatesulfate, AES) to snails (Elimia sp.) was 730 µg active AES/L, based on growth (weight gain) in a flow-through model ecosystem study. The result is based on measured concentration.
Executive summary:

A chronic toxicity test of C14 -15EO2.25S (Alkylethoxylatesulfate, AES) was conducted with Elimia sp.(snails). The growth of snails was determined after 8 weeks in the tail pools of flow-through model stream mesocosms at the P&G Experimental Stream Facility (ESF). River water was collected from the Lower East Fork of the Little Miami River, a high quality stream in Clermont County, Ohio.

The nominal test concentrations were 0, 12, 37, 111, 333 and 1000 µg active AES/L. Measured concentrations were 0, 14, 27, 75, 234 and 719 µg active AES/L. All results are based on the measured concentration of the test substance.

Mortality was 0 -10% at all exposure levels. Survival was not significantly affected at any test concentration.

Snail weight increased by 54.7 - 94.9 mg wet weight irrespective of concentration of AES. Growth was not significantly affected at any test concentration.

The No Observed Effect Concentration (NOEC) was 730 µg active AES/L, based on survival and growth (weight gain). The NOEC was the highest concentration tested.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: only short abstract available
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 202, Part II
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
not specified
Test organisms (species):
Daphnia magna
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Details on test conditions:
TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.72 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: mortality, number of young born, time of the first production of young, signs of intoxication observed

LOEC = 2.2 mg a.i./L

Description of key information

In the key-study a NOEC for invertebrates of 0.27 mg/L for reproduction could be determined after 21 days of exposure. Nevertheless for the PNEC-calculation NOEC-values were determined on the basis of a QSAR-model based on chronic risk to invertebrates. The NOEC used for PNEC-calculation is determined to be 1.2 mg/L.

Key value for chemical safety assessment

Additional information

One chronic invertebrate study is available for AES (C12-14, 1-2.5EO) Na (CAS 68891-38-3). In that study (P&G, 1977a), equivalent to the OECD guideline 211, a NOEC for surival and reproduction was determined to be 0.27 mg/L after 21 days. That reliable study is determined to be the key study. The results of that key study could be confirmed by two reliable tests conducted with comparable mixtures, alcohol C14 -15 ethoxysulfates (P&G, 1987 and P&G, 1997) resulting in NOEC-values of 0.18 mg/L (after 21 days) and 0.730 mg/L (after 8 weeks). These values are within the same range as the NOEC described by (P&G, 1977a). In addition, a further test conducted with alcohol C12 -14 ethoxysulfates available which reportes an NOEC-value of 0.72 mg/L (BASF, 1998b). That study could not be fully evaluated as only a short summary is available.