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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:
April 29 - May 6, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA-600/489/001: Short term methods for measuring the chronic toxicity of effluents and receiving waters to freshwater organisms. Deviations, reliability and validity evaluated against USEPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test).
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all exposure concentrations on days 1, 4 and 7.
- Sampling method: 50 mL of sample water was removed from each of the replicate stream channels and combined to yield a 100 mL sample for each test concentration. Each sample was treated to three successive MBAS extractions using HPLC grade chloroform. Extracted material was combined and brought to 50 mL with fresh chloroform.
- Sample storage conditions before analysis: Not available
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test material stock solutions were prepared by diluting surfactants in distilled water. Stock mixtures of the test substance were made up in 4-L batches and placed on magnetic stirrers.
- Controls: in dilution water (no test substance)
- Evidence of undissolved material: slight precipitate
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Ceriodaphnia dubia
- Strain/clone: Not available
- Justification for species other than prescribed by test guideline: Not applicable
- Source: Virginia Polytechnic Institute and State University In-house breeding culture
- Age of parental stock: Not available
- Feeding during test: Yes
- Food type: trialgal diet containing equal portions of Chlamydomonas reinhardtii, Chlamydomonas vulgaris and Selenastrum capricornutum
- Amount: Not available
- Frequency: daily at each renewal


ACCLIMATION
- Acclimation period: Not available
- Acclimation conditions (same as test or not): Not available
- Type and amount of food: trialgal diet containing equal portions of Chlamydomonas reinhardtii, Chlamydomonas vulgaris and Selenastrum capricornutum
- Feeding frequency: Not available
- Health during acclimation (any mortality observed): Not available


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: not provided
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
Not applicable
Hardness:
Not available
Test temperature:
24.9 - 25.3 ºC
pH:
Not available
Dissolved oxygen:
Not available
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations were 0, 0.05, 0.10, 0.50, 1.0 and 2.0 mg/L
Measured concentrations were 0, 0.02, 0.08, 0.20, 0.80, 1.29 mg/L
The measured concentrations were 40, 80, 40, 80 and 65 % of the nominal concentrations, respectively. Results are based on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material: Test chamber is a 35 mL disposable polystyrene cup fitted with 120-µM Nitex mesh on opposite sides
- Aeration: no (test was flow-through)
- Type of flow-through: peristaltic
- Renewal rate of test solution (frequency/flow rate): 360 mL/h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 daphnid/35 mL


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as described by Lauth et al (1990)
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: Not available
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: Not available
- Conductivity: Not available
- Salinity: Not available
- Culture medium different from test medium: Not available
- Intervals of water quality measurement: days 1,4 and 7 for each exposure concentration


OTHER TEST CONDITIONS
- Adjustment of pH: Not available
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 150 - 400 foot candles


EFFECT PARAMETERS MEASURED: mortality and number of young were assessed daily


RANGE-FINDING STUDY
- Test concentrations: 0-0.38 mg/L (see section 6.1.3., acute test, for details)
- Results used to determine the conditions for the definitive study: Yes, the results of the acute toxicity test were used to establish test concentrations for the chronic test.
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.204 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
7 d
Dose descriptor:
other: IC20
Effect conc.:
0.38 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
not specified
Remarks on result:
other: 0.25 - 0.45
Details on results:
- Mortality of parent animals: See section below (Remarks on results including tables and figures)
- No. of offspring produced per day per female: See section below (Remarks on results including tables and figures)
- Body length and weight of parent animals: Not available
- Type and number of morphological abnormalities: Not available
- Type and number of behavioural abnormalities: Not available
- Number of males and females (parental): Not available
- Time to first brood release or time to hatch: Not available
- Egg development time: Not available
- Brood size: Not available
- Time to sexual maturity: Not available
- Type and magnitude of biochemical changes: Not available
- Other biological observations: Not available
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
One-way analysis of variance was used to determine the NOEC and LOEC effect concentrations. The EC20 was determined using a nonlinear regression approach as described by Bruce and Versteeg (1992). All analyses of variance were conducted using Minitab (1993).

Chronic Effects of C16AS on Ceriodaphnia dubia (7 days; study 66165)

Actual Concentration (mg/L) % Mortality Neonates per Female S.D.
0 10 22.8 4.3
0.02 0 26.5 3.9
0.08 0 23.6 3.2
0.20 0 25.1 1.5
0.80 0 11.8* 4.7
1.29 10 4.7* 2.9

*indicates a significant difference from control

C16AS = C16 -alkyl sulfate

Validity criteria fulfilled:
yes
Remarks:
80% or greater survival in controls, 15 or more young per surviving female
Conclusions:
The 7-day NOEC of C16AS (C16-alkyl sulfate) to Ceriodaphnia dubia was 0.20 mg/L, based on measured concentrations of test substance.
Executive summary:

In a 7-day chronic toxicity study, Ceriodaphnia dubia were exposed to C16AS (C16-alkyl sulfate) at nominal concentrations of 0, 0.05, 0.10, 0.50, 1.0 and 2.0 mg/L, and measured concentrations of 0, 0.02, 0.08, 0.20, 0.80 and 1.29 mg/L under flow-through conditions. The 7-day NOEC based on measured concentrations was 0.20 mg/L, based on the most sensitive endpoint (reproduction). The toxicity study is classified as acceptable and satisfies the validity critieria as stated in the study protocol.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From Aug. 07, 1986 to Aug. 28, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Remarks:
according to EPA principles of GLP
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Weekly from two replicate vessels of each test concentration and control (0, 0.19, 0.38, 0.75, 1.5 and 3.0 mg active/L).
- Sampling method: Not available
- Sample storage conditions before analysis: The samples were preserved with 1% formalin and refrigerated until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Fresh stock solutions of 0.117 mg A.I./mL were prepared on alternating days by diluting 0.636 g of test material with distilled water to a total volume of 4000 mL. A 200 mL proportional diluter (constructed entirely of glass and silicone tubing, stoppers and sealant) calibrated to provide 50% dilutions between adjacent concentrations was used to deliver the test solution to the test vessels. The diluter was equipped with a 4500 mL glass Marriotte bottle/tube siphon delivery system which introduced 10 mL of the test material stock solution (0.117 mg A.I./mL) into 380 mL of dilution water in the diluter mixing chamber during each diluter cycle. This 390 mL solution (3.0 mg A. I. /L) served as the highest treatment level from which calibrated volumes were diluted to provide the 50% nominal concentration gradient (0.19 to 3 mg/L of test solution). Five cm lengths of 1 mm (inside diameter) glass capillary tubing were inserted through silicone stoppers in the mixing/splitting chambers of the diluter and into the test solution delivery tubes. This tubing served to restrict the flow of the test solutions, minimizing potentially stressful turbulence in the test vessels and providing equal distribution of the test solutions to the replicate vessels. A complete check of diluter function was made daily.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Not reported
- Source: D. magna were obtained from populations cultured at Sprinborn Life Sciences maintained at Bionomics Aquatic Toxicology Laboratory
- Maintenance of stock culture: Daphnids were cultured in 57 L glass aquaria containing 40 L of water. Water used to culture the daphnids was prepared in the same manner and had the same characteristics as described for diluent water. The cultures were aerated sufficiently to maintain the dissolved oxygen concentration above 65 percent of saturation. Culture water was maintained at 22 (+/-2) °C. Culture organisms were fed daily a combination of Rangen trout food and unicellular green algae. Each culture aquarium was cleaned within a 14 d period.
- Age of parental stock : Neonate (< 24 h old)
- Feeding during test: Yes
- Food type: Suspension of Fleishmann's yeast (5 mg/mL) and a suspension of green algae (Ankistrodesmus falcatus; 4.0x10 (7) cells/mL).
- Amount: 2 mL of yeast suspension and 1 mL of algal suspension
- Frequency: Three times daily on weekdays (Monday to Friday) and twice daily on weekends and holidays.

ACCLIMATION
- Acclimation period: Daphnids were cultured and tested under the same conditions.
- Acclimation conditions: Same as test
- Type and amount of food: See description of culture above.
- Feeding frequency: See description of culture above.
- Health during acclimation (any mortality observed): Not reported


Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
150-160 mg/L (as CaCO3)
Test temperature:
19 to 20°C (Brooklyn alcohol thermometer)
pH:
7.8-8.3 (Instrumentation Laboratory Model #175 pH meter)
Dissolved oxygen:
7.3 - 8.1 mg/L (YSI Model #57 dissolved oxygen meter)
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal: 0, 0.19, 0.38, 0.75, 1.5 and 3.0 mg active/L
Mean Measured concentrations: 0, 0.03, 0.05, 0.14, 0.48 and 1.7 mg active/L. Mean measured concentrations were 13-57% of nominal, and results are based on mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass battery jars
- Type: Open
- Material, size, headspace, fill volume: Glass battery jars with volume capacity of 1.8 L containing 1.75 L test media. The jars had 3.5 x 8.0 cm notch cut on the upper edge for facilitating test solution drainage from the test vessels. The notch was covered with nitex 40 mesh screen to prevent loss of daphnids.
- Aeration: No (test was flow-through)
- Type of flow-through: Proportional diluter
- Renewal rate of test solution (frequency/flow rate): 4.5 aquarium volumes per 24 h period (to provide a 90% test solution replacement rate of approx. 9 h)
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four
- Biomass loading rate: There were 20 daphnids in each aquaria containing 1.75 L test media according to the study report. Loading rate was therefore 1 daphnid/87.5 mL test solution.
- Others: Test vessels were brushed to remove algal growth and the solution filtered through a fine mesh net a minimum of twice each wk

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Diluent water was 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: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: 110-120 (mg/L CaCO3)
- Ca/mg ratio: Not reported
- Specific conductivity: 440-460 µmhos/cm (measured by YSI Model 33 conductivity-salinity meter)
- Salinity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: The following parameters were observed in one aquarium from each treatment and the control solution: Temperature: measured daily; Dissolved oxygen: measured every; Total hardness, alkalinity, specific conductivity and pH: monitored weekly
- Filtration: Dilution water is filtered through an amberlite XAD-7 resin column prior to delivery to the diluter to remove any potential organic contaminants.

OTHER TEST CONDITIONS
- Adjustment of pH: Not reported
- Photoperiod: 16 h light and 8 h darkness
- Light intensity: 15 to 100 foot candles using Cool White and Gro-Lux fluorescent lights (measured with a General Electric type 214 light meter)

EFFECT PARAMETERS MEASURED: Adult survival and measurements of offspring production were made on Day 4 and every weekday, from Day 7 through 21. The offspring were removed counted and discarded.

RANGE-FINDING STUDY: Yes. A flow-through acute toxicity test was conducted at Springborn Life Sciences for determining the concentration for chronic exposure.
- Test concentrations: Daphnids were exposed to nominal concentration ranging from 0.19 to 3.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. After 24 h exposure, 50% of the daphnids died at concentrations ≥ 1.5 mg active/L while no toxicant related mortalities or adverse effects were observed at concentration ≤ 0.75 mg/L. Based on this data, the concentrations were selected for the chronic exposure.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: -
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: reproduction and mortality
Remarks on result:
other: -
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
0.084 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: reproduction and mortality
Remarks on result:
other: Geometric mean of >0.050 and <0.14 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: -
Details on results:
- Mortality of parent animals: No daphnids survived the 21 d exposure at the highest test concentration (1.7 mg/L). Survival of daphnids in test concentrations of ≤0.48 mg/L ranged from 71 to 90% which was not significantly different from the control survival (85%). In two replicates of the 0.050 mg/L, 100% mortality was observed in both solutions by Day 20 while no mortality was observed in the remaining two replicates of this treatment level or in all replicates of the next two higher test concentrations (0.14 and 0.48 mg/L), Therefore, it was believed that the mortality was due to replicate solution contamination on test Day 18. Hence, mean survival for this treatment level was based on the remaining two replicates where survival was 80 and 85% respectively.
For details, please refer ‘Table 1’ under 'Any other information on results incl. table' section.
- No. of offspring produced: During the 21 d exposure, daphnids exposed to 0.48 and 0.14 mg/L produced an average of 0 and 24 offspring per female respectively, which was significantly less (p ≤ 0.05) than the average number of offspring produced by control organisms. Exposure at ≤ 0.050 mg/L did not affect the reproductive activity of the test organisms. Since the survival of daphnids was significantly reduced in the highest concentration, reproduction data for this treatment level was not compared to reproduction of the control organisms Control daphnids produced an average of 86 ±7 offspring by test termination.
For details, please refer ‘Table 2’ under 'Any other information on results incl. table' section.
- Body length and weight of parent animals: Not reported
- Type and number of morphological abnormalities: Not reported
- Type and number of behavioural abnormalities: Not reported
- Number of males and females (parental): Not reported
- Time to first brood release or time to hatch: Day 8 in control
- Egg development time: Not reported
- Brood size: Not reported
- Time to sexual maturity: Not reported
- Type and magnitude of biochemical changes: Not reported
- Other biological observations: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No
- Others: Throughout the 21 d exposure period no visible sign of insoluble material was observed in any of the test solutions
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Mortality data transformed to arc sin percentage and the determinations of cumulative production of offspring per female were analyzed by 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.

Table 1. Weekly mean percentage survival of daphnia during Chronic exposure to C14-15 Sodium alcohol sulphate (Study # 34655)a

Mean Measured Concentration (mg/L)

Mean Percentage Survival (Standard Deviation)

Day 7

Day 14

Day 21

Control

100 (0)

94 (6)

85 (14)

0.03

99 (3)

88 (12)

80 (14)

0.05a

98 (4 )

93 (11)

83 (4)

0.14

100 (0)

96 (5)

90 (11)

0.48

95 (4)

89 (5)

71 (23)

1.7

0b(0)

0 (0)

0c(0)

aMean percentage survival calculations for the concentration are based on two replicates (replicates A and B) as it appears that test system contamination is responsible for 0% survival in replicates C and D. It is believed that this mortality was not toxicant related.

bAll daphnids exposed to this treatment level died within the initial 7 d of the chronic study.

cSignificantly (P≤0.05) different from the survival of the control

Table 2. Cumulative number of offspring produced per female Daphnia magna during the 21d chronic exposure to C14-15 Sodium alcohol sulphate (Study # 34655)

Mean Measured Concentration (mg/L)

Mean (Standard Deviation) Cumulative number of offspring/female

Day 7

Day 8

Day 11

Day 12

Day 13

Day 14

Day 15

Day 18

Day 19

Day 20

Day 21

Control

0 (1)

4 (2)

22 (5)

30 (6)

37 (4)

49 (7)

52 (7)

66 (8)

72 (6)

78 (6)

86 (7)

0.03

1 (1)

5 (2)

26 (7)

35 (10)

44 (11)

52 (11)

55 (13)

69 (14)

75 (15)

83 (18)

87 (16)

0.050c

1 (1)

3 (3)

23 (12)

28(16)

37 (21)

44 (20)

47 (20)

59 (16)

63 (17)

67 (17)

85 (28)

0.14

0 (0)

1 (1)

10 (5)

12 (7)

13 (8)

16 (10)

19 (12)

22 (10)

23 (9)

24 (9)

24a(7)

0.48

0(0)

0(0)

0 (0)

0(0)

0(0)

0 (0)

0(0)

0 (0)

0(0)

0(0)

0a(0)

1.7

b

-

-

-

-

-

-

-

-

-

-

aSignificantly different (p ≤ 0.05) from control reproduction.

bNo surviving adult daphnids.

cCalculations based on replicates A and B only, as non-toxicant related mortality was noted in replicates C and D of this treatment level.

Statistical analysis performed on test Day 21 data only.

Validity criteria fulfilled:
yes
Conclusions:
The 21 d NOEC of C14-15 Sodium alcohol sulphate to Daphnia magna was 0.050 mg/L, based on reproduction and mortality. The result is based on measured concentration.
Executive summary:

The 21 -d chronic toxicity test of C14-15 Sodium alcohol sulphate (C45AS) to Daphnia magna was determined under flow-through condition, following the OECD 211 guideline. The mean measured concentrations were 0.0, 0.03, 0.05, 0.14, 0.48 and 1.7 mg/L of active ingredient. There were 4 replicates at each test concentration and control level.

At the test termination, no daphnids survived exposure to the highest test concentration. Survival of daphnids at concentrations of ≤0.48 mg/L ranged from 71 to 90%, which were statistically comparable to the survival of control daphnids (85%). Reproduction among daphnids exposed to 0.48 mg/L and 0.14 mg/L was significantly less (average of 0 and 24 offspring per female, respectively) than that of the control daphnids (86). Exposure to concentrations ≤0.050 mg/L did not affect the reproduction of the daphnids.

Hence, the NOEC, based on mortality and reproduction was 0.050 mg/L (based on measured concentrations).

This Chronic toxicity test is classified as acceptable, and satisfies the guideline requirements for the OECD 211.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From Oct. 3, 1991 to Oct. 24, 1991
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
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.19, 0.38, 0.75, 1.5, and 3 mg a.i./L
- Sampling frequency: Prior to the start of the definitive exposure, and on Day 0, 7, 15 and 21 of the test, samples were collected for analysis of test substance by MBAS. Samples were also collected on Day 0, 7, 15, 20 and 21 for analysis of test solution for C14 and C15 alcohols by GC. At each sampling interval during the in-life phase, two of the four replicates per concentration and control were sampled, alternating between replicates A and B or C and D at each sampling interval.
- Sampling method: Samples were collected from approx. midpoint of the test vessel by volumetric pipet. For MBAS analysis, 50 mL samples were collected from the 1 and 3 mg/L test solutions, and 100 mL samples from the 1.5 and 0.75 mg/L test solutions, using volumetric pipet. 250 mL samples were siphoned into graduated cylinder from the 0.38, 0.15, and control test solutions. For fatty alcohol analysis, 250 mL samples were collected from each test solution. All collected samples were stabilized with formalin.
Details are provided in the study report.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: The test material was heated and stirred at 60°C for approx. 3 h prior to weighing. A diluter stock solution of 1.44 mg a.i./mL was prepared weekly by diluting 13 g (2.89 g active ingredient) test material with distilled water to a volume of 2 L. This 2 L stock solution was stirred for approx. 24 h before use.
- Evidence of undissolved material: No. Diluter stocks were observed to be a cloudy white, homogeneous solution. Each solution was stirred continuously during delivery to the test system.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: In house (population cultured at Springborn laboratories, Inc.)
- Maintenance of stock culture: Daphnids were cultured under static renewal conditions (20 ±2°C) in well water fortified to a total hardness of approx. 160-180 mg/L CaCO3. The daphnids culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness (intensity: 30-50 footcandles). Daphnids were fed daily a combination of green algae (Ankistrodesmus falcatus) and a trout food suspension. Food sources were considered acceptable since the total concentration of pesticides was <0.3 mg/kg.
- Age at test begin: ≤24h old
- Feeding during test: Yes
- Food type: Diet consisting of a suspension of trout food suspension (5 mg/L), a suspension of green algae (Ankistrodesmus falcatus; 4 x 10 (7) cells/mL) and Selco (a commercial mixture of protein and fatty acids, 0.6 mg/mL). The food was introduced at a rate of 2 mL of trout food suspension, 3 mL of algal suspension and 0.5 mL Selco food supplement.
- Frequency: Three times daily on weekdays and twice daily on weekends.

ACCLIMATION
- Acclimation period: Not reported. However, daphnids were obtained in-house, cultured under the same conditions as the test
- Acclimation conditions: Same as test
- Type and amount of food: See description of culture above.
- Feeding frequency: See description of culture above.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
Total hardness range: 170-180 mg/L CaCO3
Test temperature:
19 ±0.5 °C
pH:
pH range: 7.8-8.2
Dissolved oxygen:
Dissolved oxygen range: 7.9-8.7
Nominal and measured concentrations:
Nominal: 0, 0.19, 0.38, 0.75. 1.5, and 3 mg a.i./L
Mean measured concentrations: 0.091, 0.15, 0.31, 0.63, and 1.2 mg a.i./L. Mean measured concentrations were 39-48% of nominal, and results are based on mean measured concentrations.
(Note: Analysis of solutions during pretest period of the definitive chronic exposure showed measured concentrations averaged 60% of nominal.)
Details on test conditions:
TEST SYSTEM
- Test vessel: Jars
-Type: Open (loosely covered with plastic wrap throughout the duration exposure period.
- Material: 1.4L glass battery jars. Test vessels were washed a minimum of three times per week during the exposure period.
- Aeration: No, but the test design was flow-through.
- Type of flow-through: Continuous flow serial diluter
- Renewal rate of test solution: Test solution were delivered to the test vessels at an approx. rate of 6.6 aquarium volumes per 24-h period in order to provide a 90% test solution replacement rate of approximately 6 h.
- No. of organisms per vessel: 10
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- Biomass loading rate: Not specified (approx. 1 organism/100 ml)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water was prepared in 1900 L batches by fortifying well water according to the formula for hard water (ASTM, 1990), and filtering through an Amberlite XAD-7 resin column and a carbon filter to remove any potential organic contaminates.
- Frequency of preparation of dilution water: Generally several batches of water were prepared each week. The frequency at which the dilution water prepared depended on the requirements of the laboratory. Fortified water was discarded if not used within 14 days of preparation.
- Total alkalinity: 110-120 mg/L CaCO3
- Specific conductivity: 500 µmhos/cm
- Pesticides: Pesticides and PCBs were not detected at concentrations that were considered toxic in any of the water sample analyzed.
- Total organic carbon: 0.89 mg/L (as determined in the month of October 1991)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured daily in one replicate vessel of each treatment level and control. In addition, temperature was continuously monitored in one replicate of the 3 mg a.i./L solution throughout the study. DO 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 were monitored weekly in one replicate vessel from each treatment level and the control solution. The pH, dissolved oxy gen concentration and temperature were also measured once a week in all replicate vessels of each treatment level and the control.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Illuminated with Durotest Cool-White and Durotest vita-Lite fluorescent light at an intensity of 30 to 50 footcandles

EFFECT PARAMETERS MEASURED: Adult survival and measurements of offspring production were made on Days 1, 2, 4, 7, 8, 11, 14, 15, 18 and 21. The offspring were removed, counted and discarded. Abnormal behavior and appearance was also noted.

RANGE-FINDING STUDY: Yes, test substance concentration for 21 days chronic exposure were selected based on the results of preliminary flow-through testing conducted at SLI. Daphnids ≤24 h old were exposed under flow-through conditions.
- Test concentrations: 0.19, 0.38, 0.75, 1.5, and 3 mg a.i./L
- Results used to determine the conditions for the definitive study: Yes. Following 10 days of exposure, 100% survival was observed in all treatment levels tested. Reproduction averaged 0 offspring per female in the highest treatment level tested. All the daphnids in the highest treatment tested were visibly smaller in size comparing to control daphnids. Reproduction among daphnids exposed to the remaining nominal test concentration (0.19-1.5 mg a.i./L) ranged from 13-22 offspring per female. Reproduction among control daphnids averaged 18 offspring/female.
Reference substance (positive control):
not required
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: reproduction and survival
Details on results:
- Mortality of parent animals: Survival of daphnids exposed to the treatment levels tested (0.091-1.2 mg a.i./L) ranged from 95-98% and was comparable to the survival of the daphnids exposed to the control solution (met the minimum standard criteria established by USEPA i.e. ≥80% survival)
- No. of offspring produced: By test termination, control daphnids had produced an average of 234 offspring. The mean reproduction of daphnids exposed to 0.091-1.2 mg a.i./L ranged from 238-254 offspring per female which was statistically comparable to the control (met the minimum standard criteria established by USEPA i.e. 50 offspring/female)
- Time to first brood release or time to hatch: Control daphnids began releasing offspring by test Day 7. Release of first brood offspring by daphnids exposed to treatment solutions ≤1.2 mg a.i./L occurred by test Day 7 and was not adversely affected by the concentration of test substance tested.
- Other biological observations: No adverse sublethal effects were observed among daphnids at all treatment levels.
- Effect concentrations exceeding solubility of substance in test medium: No
Further details are provided in the study report.
Reported statistics and error estimates:
- Significant differences in the percentage survival were determined after transformation (e.g. arcsine square root percentage) of the data.
- The Shapiro Wilks test for normality was conducted and compared the observed sample distribution with a normal distribution. The assumption that observations were normally distributed was validated before subsequent analysis, following parametric procedures, was performed. If the data was not normally distributed, then a non-parametric procedure was used for subsequent analysis.
- As a check on the assumptions of homogeneity of variance implicit in parametric statistics, data for each endpoint were analyzed using Bartlett’s test.
- Survival data were analyzed prior to the analysis of reproduction data; if there was a concentration that caused significant adverse effects on survival, then that level was excluded from the analysis of reproduction data.

Table 1: Survival of Parent daphnids during 21-day chronic exposure (study # 35210)

Mean measured concentration mg a.i./L

Mean % survival

 Day 1

 Day 2

 Day 4

 Day 7

 Day 8

 Day 11

 Day 14

 Day 15

   Day 18

 Day 21

1.2

95

95

95

951

95

952

95

95

95

95

0.63

98

98

982

952

95

95

95

95

95

95

0.31

100

100

100

98

98

98

98

98

98

98

0.15

100

100

100

1003

98

98

98

98

98

98

0.091

984

98

98

98

98

98

98

98

98

98

Control

100

100

98

98

98

98

98

98

98

95

Flow-through test of C45 alkyl sulfate (CAS # 68890-70-0) to Daphnia magna.

121% daphnids were observed to be swimming, carrying organic matter

2daphnids were observed to be pale; 26% at Day 11 (test concentration 1.2 a.i./L); 3% and 5% at Day 4 and Day 7 respectively (test concentration 0.63 a.i./L)

33% daphnids were observed to be small and pale

4daphnids were observed to be on the surface of the test solution

Table 2: Total reproduction per daphnid during the 21-day chronic exposure to alkyl sulfate (study # 35210)

Mean measured concentration (mg a.i./L)

Mean cumulative number of offspring /female

 Day 7

 Day 8

 Day 11

 Day 14

 Day 15

   Day 18

 Day 21

1.2

4

5

36

83

85

162

239

0.63

3

8

36

81

84

164

238

0.31

4

8

41

87

87

167

252

0.15

3

8

39

90

92

174

254

0.091

2

8

33

81

85

185

243

Control

0

6

31

77

81

159

234

Flow-through test of C45 alkyl sulfate (CAS # 68890-70-0).

Validity criteria fulfilled:
yes
Conclusions:
The 21 d NOEC of C14-15 Alkyl Sulfate to Daphnia magna was 1.2 mg/L based on reproduction and adult mortality. The result is based on mean measured concentration.
Executive summary:

The 21 d chronic toxicity of C14-15 Alkyl Sulfate to Daphnia magna was determined under flow-through conditions, following the OECD 211 guideline. The test substance was comprised of C14 alkyl chainlength (44%) and C15 alkyl chainlength (56%). The test was conducted in well water, 175 mg/L hardness, at 19°C.

There were 4 replicates (10 daphnids each) at each test concentration and the control. The mean measured test substance concentrations were 0, 0.091, 0.15, 0.31, 0.63 and 1.2 mg a.i./L (39-48% of nominal).

There were no significant effects on daphnid survival or reproduction at any test concentration in this test. At test termination, mean reproduction of daphnids exposed to alkyl sulfate (0.091-1.2 mg/L) ranged from 238-254 offspring per female which was statistically comparable to the control (234 offspring/female). Survival of daphnids ranged from 95-98% at all treatment levels and was comparable to the survival of the daphnids exposed to the control solution.

Hence, the NOEC, based on survival and reproduction was1.2 mg active/L. The result is based on mean measured concentration.

This chronic toxicity test is classified as acceptable, and satisfies the guideline requirements for the OECD 211.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 - 23 March 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA-600/489/001: Short term methods for measuring the chronic toxicity of effluents and receiving waters to freshwater organisms. Deviations, reliability and validity evaluated against USEPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test).
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all exposure concentrations on days 1, 4 and 7.
- Sampling method: 50 mL of sample water was removed from each of the replicate stream channels and combined to yield a 100 mL sample for each test concentration. Each sample was treated to three successive MBAS extractions using HPLC grade chloroform. Extracted material was combined and brought to 50 mL with fresh chloroform.
- Sample storage conditions before analysis: Not available
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test material stock solutions were prepared by diluting surfactants in distilled water. Stock mixtures of the test substance were made up in 4-L batches and placed on magnetic stirrers.
- Controls: in dilution water (without test substance)
- Evidence of undissolved material: none
Test organisms (species):
Ceriodaphnia dubia
Details on test organisms:
TEST ORGANISM
- Common name: Ceriodaphnia dubia
- Strain/clone: Not available
- Justification for species other than prescribed by test guideline: Not applicable
- Source: Virginia Polytechnic Institute and State University In-house breeding culture
- Feeding during test: Yes
- Food type: trialgal diet containing equal portions of Chlamydomonas reinhardtii, Chlamydomonas vulgaris and Selenastrum capricornutum
- Frequency: daily at each renewal

ACCLIMATION
- Type and amount of food: trialgal diet containing equal portions of Chlamydomonas reinhardtii, Chlamydomonas vulgaris and Selenastrum capricornutum.
- Feeding frequency: Not available
- Health during acclimation (any mortality observed): Not available

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: not provided
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
Not applicable
Hardness:
Not available
Test temperature:
24.9 - 25.3 ºC
pH:
Not available
Dissolved oxygen:
Not available
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations were 0, 0.10, 0.25, 0.50, 1.0 and 2.50 mg/L
Measured concentrations were 0, 0.05, 0.25, 0.39, 1.73 and 2.53 mg/L
The measured concentrations were 50, 100, 78, 173 and 101% of the nominal concentrations, respectively. Results are based on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material: Test chamber is a 35 mL disposable polystyrene cup fitted with 120-µM Nitex mesh on opposite sides
- Aeration: no (test was flow-through)
- Type of flow-through: peristaltic
- Renewal rate of test solution (frequency/flow rate): 360 mL/h
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 daphnid/35 mL


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as described by Lauth et al (1990)
- Intervals of water quality measurement: days 1,4 and 7 for each exposure concentration


OTHER TEST CONDITIONS
- Adjustment of pH: Not available
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 150 - 400 foot candles


EFFECT PARAMETERS MEASURED: mortality and number of young were assessed daily


RANGE-FINDING STUDY
- Test concentrations: 0-6.38 mg/L (see section 6.1.3., acute test, for details)
- Results used to determine the conditions for the definitive study: Yes, the results of the acute toxicity test was used to establish test concentrations for the chronic test.
Reference substance (positive control):
no
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.05 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.05 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
7 d
Dose descriptor:
other: IC20
Effect conc.:
0.18 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
not specified
Remarks on result:
other: 0.04 - 0.32
Details on results:
- Mortality of parent animals: See section below (Remarks on results including tables and figures)
- No. of offspring produced per day per female: See section below (Remarks on results including tables and figures)
- Body length and weight of parent animals: Not available
- Type and number of morphological abnormalities: Not available
- Type and number of behavioural abnormalities: Not available
- Number of males and females (parental): Not available
- Time to first brood release or time to hatch: Not available
- Egg development time: Not available
- Brood size: not available
- Time to sexual maturity: Not available
- Type and magnitude of biochemical changes: Not available
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
One-way analysis of variance was used to determine the NOEC and LOEC effect concentrations. The EC20 was determined using a nonlinear regression approach as described by Bruce and Versteeg (1992). All analyses of variance were conducted using Minitab (1993).

Chronic Effects of C14AS on Ceriodaphnia dubia (7 days; study 66165)

Actual Concentration (mg/L) % Mortality Neonates per Female S.D.
0 0 25.3 4.5
0.05 0 20.5* 3.2
0.25 0 19.4* 2.0
0.39 0 15.9* 3.4
1.73 0 6.4* 3.1
2.5360 60 0* 0

*indicates a significant difference from control

C14AS = C14 -alkyl sulfate

Validity criteria fulfilled:
yes
Remarks:
80% or greater survival in controls, 15 or more young per surviving female
Conclusions:
The 7-day NOEC of C14AS (C14-alkyl sulfate) to Ceriodaphnia dubia was <0.05 mg/L, based on measured concentrations of test substance.
Executive summary:

In a 7-day chronic toxicity study, Ceriodaphnia dubia were exposed to C14AS (C14-alkyl sulfate) at nominal concentrations of 0, 0.10, 0.25, 0.50, 1.0 and 2.50 mg/L, and measured concentrations of 0, 0.05, 0.25, 0.39, 1.73, and 2.53 mg/L under flow-through conditions. The 7-day NOEC based on measured concentrations was <0.05 mg/L, based on the most sensitive endpoint (reproduction). The toxicity study is classified as acceptable and satisfies the validity critieria as stated in the study protocol.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
ummary of available data used for the endpoint assessment of the target substance
Adequacy of study:
key study
Justification for type of information:
Refer to the Category Approach Justification document provided in IUCLID6 Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.05 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
Source, key, 1191-50-1, 1995a
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.05 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
Source, key, 1191-50-1, 1995a
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.204 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other:
Remarks:
Source, key, 1120-01-0, 1995b
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
Source, key, 1120-01-0, 1995b

Description of key information

7d NOEC (C. dubia, reproduction) = 0.072 mg/L (measured concentration, EPA-600/489/001)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.072 mg/L

Additional information

Since no reliable chronic test involving aquatic invertebrates is available for C14-16AS Na (CAS 91648-54-3), in accordance to Regulation (EC) No. 1907/2006 Annex XI, 1.5, a read-across to another structurally related category members was conducted. Read-across data from the following alkyl sulfates were used: C14AS Na (CAS 1191-50-0), C16AS Na (CAS 1120-01-0) and C14-15AS Na.

A non GLP, 7-day chronic toxicity study with C14AS (CAS 1191-50-0) was performed according EPA-600/489/001 guideline (P&G 1995a). Ceriodaphnia dubia were exposed at measured concentrations of 0, 0.05, 0.25, 0.39, 1.73, and 2.53 mg/L under flow-through conditions. The 7-day NOEC based on measured concentrations was 0.05 mg/L, based on the most sensitive endpoint (reproduction).

Another non-GLP, 7-day reproduction toxicity test with Ceriodaphnia dubia performed according to EPA-600/489/001 guideline is available for C16AS Na (CAS 1120-01-0, P&G 1995b). Daphnids were exposed under flow-through conditions to five test concentrations of 0, 0.02, 0.08, 0.20, 0.80 and 1.29 mg/L (measured concentrations). Mortality and reproduction were evaluated daily. The NOEC value for reproduction determined after 7 days of exposure to test substance was 0.204 mg/L (measured concentration).

The 21-d chronic toxicity of C14-15AS Na to Daphnia magna was determined under flow-through conditions, following the OECD 211 guideline (P&G 1992). The test substance was comprised of C14 alkyl chainlength (44%) and C15 alkyl chainlength (56%). The mean measured test substance concentrations were 0, 0.091, 0.15, 0.31, 0.63 and 1.2 mg a.i./L (39-48% of nominal). There were no significant effects on daphnids survival or reproduction at any test concentration in this test. At test termination, mean reproduction of daphnids exposed to alkyl sulfate (0.091-1.2 mg/L) ranged from 238-254 offspring per female which was statistically comparable to the control (234 offspring/female). Survival of daphnids ranged from 95-98% at all treatment levels and was comparable to the survival of the daphnids exposed to the control solution. The NOEC, based on survival and reproduction was1.2 mg a.i./L (mean measured concentration).

Finally, another 21-d chronic toxicity test of C14-15AS Na to Daphnia magna was performed under flow-through condition, following the OECD 211 guideline (P&G 1987). The mean measured concentrations were 0.0, 0.03, 0.05, 0.14, 0.48 and 1.7 mg/L of active ingredient. At the test termination, no daphnids survived exposure to the highest test concentration. Survival of daphnids at concentrations of ≤0.48 mg/L ranged from 71 to 90%, which were statistically comparable to the survival of control daphnids (85%). Reproduction among daphnids exposed to 0.48 mg/L and 0.14 mg/L was significantly less (average of 0 and 24 offspring per female, respectively) than that of the control daphnids (86). Exposure to concentrations ≤0.050 mg/L did not affect the reproduction of the daphnids. The NOEC, based on mortality and reproduction was 0.050 mg/L (based on measured concentrations).

Due to the structural and property similarities with the tested category members these results can also be used to determine the long-term toxicity to aquatic invertebrates of C14-16AS Na (CAS 91648-54-3). The composition of C14-16AS Na (CAS 91648-54-3) based on the normalized mean values of provided ranges: C14: 60% and C16: 40%. The NOEC values for C14AS Na (CAS 1191-50-0) and C16AS Na (CAS 1120-01-0) to aquatic invertebrates were 0.05 mg/L and 0.204 mg/L respectively. Using the additivity formula stipulated in the EC Regulation 1272/2008, the EC50 value of C14-16AS Na (CAS 91648-54-3) is 0.072 mg/L.