Phenol, paraalkylation products with C10-15 branched olefins ( C12 rich) derived from propene oligomerization, calcium salts, sulfurized, including distillates (petroleum), hydrotreated, solvent-refined, solvent-dewaxed, or catalyc dewaxed, light or heavy paraffinic C15-C50

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiaion: October 2, 1996 Study completion: March 17, 1997, Testing phase dates November 8-12 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study following GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 130, 220, 360, 600, and 1,000 mg/L
- Sampling method: The measured concentrations of total organic carbon (TOC) were determined in samples of test media collected at the beginning and end of the toxicity test. Beginning samples were collected in duplicate from the control and the lowest and highest test concentrations prior to distribution to test vessels. At the end of the test, samples taken from each of the three replicates for the control and for the lowest and highest test concentrations were pooled together, and duplicate samples were taken from each of these three pooled solutions.
- Sample storage conditions before analysis: Samples were stored in 125 ml amber glass bottles, preserved with sulfuric acid, and placed in a refrigerator. The samples were not passed through a 0.45 pm filter prior to analysis. Samples were transferred to the analytical laboratory at Katahdin Analytical Services, Inc., in Portland, Maine for analysis.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The WAFs were prepared only at the beginning of the test. Each of the five WAFs were prepared by combining the appropriate amount of test substance and dilution water in a mixing vessel equipped with a magnetic stirrer (the vortex extended from the surface approximately 25% of the way to the bottom of the mixing vessel) and stirring these mixtures for approximately 20 hours, settling the mixtures for approximately four hours, and siphoning the water phase containing the WAF.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: UTEX 1648
- Source (laboratory, culture collection): originally procured from the Culture Collection of Algae at the University of Texas at Austin.
- Age of inoculum (at test initiation): The inoculum used to initiate the test was 7 days old.

ACCLIMATION
- Acclimation period: The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

No data

Test temperature:

23.5 to 23.7 deg C

pH:

pH: initial = 7.4 – 7.5, final = 9.2 – 10.6

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Test Levels: Control, 130, 220, 360, 600, and 1000 mg/L WAF loading rates.

Details on test conditions:

Test System: Individual water accommodated fractions (WAFs) were prepared for each test level. Test solutions were not renewed during the test. To prepare the WAFs, a measured weight of test material was added to a measured volume of dilution water and stirred for 20 hours. Stirring was
accomplished using a magnetic stir bar. Mixing speed was adjusted such that a vortex formed that reached approximately 25% of the distance to the bottom. Following the mixing period, the test solutions were allowed to stand for 4 hours before the water phase was siphoned from the mixing
vessel. The siphoned water phase (i.e., WAF) was used for the aquatic toxicity test.

Test Conditions: A static test was conducted; i.e., there was no daily renewal of test solution. Three 100-mL replicates per treatment,
inoculum ~10,000 cells/mL. The 250-mL Erlenmeyer flasks (test vessels) were covered with inverted glass beakers to reduce entry of dust, etc.
During the test all treatment and control flasks were randomly placed on an orbital shaker adjusted to approximately 100 cycles per minute under
constant light (24 hours/day). Daily cell counts were made visually by means of direct microscopic examination with a hemocytometer.Light:
Cool-white fluorescent lights provided a light intensity of 360-370 footcandles.

Measured concentrations were not determined.

Element basis (# of cells/mL) = approximately 10,000 cells/mL.

Method of calculating mean measured algal cell densities: microscopic examination using a haemocytometer.


TEST SYSTEM
- Test vessel: 250 ml glass Erlenmeyer flasks
- Type (delete if not applicable): covered with inverted glass beakers.
- Material, size, headspace, fill volume: containing 100 ml of test solution (water depth was approximately 2.5 cm)
- Initial cells density: 10,000 cells/ml
- Control end cells density: 1,453,000 cells/ml (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3


TEST MEDIUM / WATER PARAMETERS
Water used for acclimation of test organisms and for all toxicity testing was sterile enriched media (U.S. EPA, 1978; T.R. Wilbury Standard Operating Procedure number 6) adjusted to a pH of 7.5. The media had 1.2 mg/L total organic carbon and <10 mg/L total suspended solids at the beginning of the test and 2.6 mg/L total organic carbon and 27 mg/L total suspended solids at the end of the test (96 hour values result, at least in part, from the presence of algal cells).


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: A 24 hour light and 0 hour dark photoperiod was automatically maintained with cool-white fluorescent lights
- Light intensity and quality: approximately 360-370 footcandles


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The number of algal cells/ml in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculation, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a haemocytometer.

TEST CONCENTRATIONS
- Range finding study:
A range finding test was conducted from October 28 to November 1, 1996 with the WAF of three concentrations of the test material and dilution water: 10, 100, and 1,000 mg/L. The test was conducted with two replicates of each concentration., Each replicate initially contained approximately 10,000 algal cells/ml. At the conclusion of the 96 hour test, the number of cells/mL in the test vessels equaled the following percents of the number of cells/mL in the control vessels: 10 mg/L = 92%, 100 mg/L = 97%, and 1,000 mg/L = 97%.

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

360 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

840 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): The algal population grew well during the test, resulting in an average of 1,453,000 cells/ml in the control after 96 hours.
- Observation of abnormalities (for algal test): No effects (size differences, unusual cell shapes, colors, flocculation, adherence of cells to test containers, or aggregation of cells) were noted during the test.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

The average specific growth rate was calculated as the natural log of the number of cells/ml at 24, 48, 72, and 96 hours of exposure, minus the natural log of the number of cells/ml at 0 hours, divided by the hour of exposure. Results of the toxicity test were interpreted by standard statistical techniques when possible. All calculations were performed using nominal concentrations of the test material with the number of cells/ml, then with the average specific growth rates.

The probit method (Stephan, 1983) was used for calculating the 24, 72, and 96 hour effective concentrations (EC10s, EC50s, and EC90s). The probit method was used despite cautionary statements because EC50 values compared well with values calculated by other methods. The 48 hour effective concentrations could not be calculated because algal growth in all vessels was 83% or greater than control growth. The 96 hour no observed effect concentration (NOEC) was determined using TOXSTAT 3.3 (Gulley, et al., 1990) which calculated a one-way analysis of variance (ANOVA) of the number of cells/ml and of the average specific growth rate in each test vessel at the end of the test.
The data was tested for normal distribution using a Shapiro Wilks test and for homogeneity of variance using a Bartletts test.

A determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test. After 96 hours of testing, 0.5 ml of solution from each 1,000 mg/L test vessel were combined into one vessel containing 100 ml of fresh media. The resulting solution was incubated under test conditions for 72 hours. During this time, algae increased from a calculated concentration of 12,540 cells/ml to 374,000 cells/ml, indicating that the effect of the test material at the highest tested concentration was algistatic rather than algicidal.

The 96 hour EL50 (presented as “EC50” in report) for both cells per mL and growth rate is >1,000 mg/L nominal WAF loading rate. The 96 hour NOELR (presented as “NOEC” in report) is 360 mg/L nominal WAF loading rate as calculated using the number of cells per mL, and 220 mg/L nominal WAF loading rate as calculated using average specific growth rate.

Test results reported in original study as “effect concentrations (EC)” and “no observed effect concentrations (NOEC)” are reported in this summary as “effect loading (EL)” and “no observed effect loading rate (NOELR)”, respectively, because test results are based on nominal WAF loading rates.

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour EL50 (presented as “EC50” in report) for both cells per mL and growth rate is >1,000 mg/L nominal WAF loading rate. The 96 hour NOELR (presented as “NOEC” in report) is 360 mg/L nominal WAF loading rate as calculated using the number of cells per mL, and 220 mg/L nominal WAF loading rate as calculated using average specific growth rate.

Executive summary:

The acute toxicity of the water accommodated fraction (WAF) of five mixtures of the test material and water to the freshwater alga, Selenastrum capricornutum, was investigated during a study conducted at T.R. Wilbury Laboratories, Inc. The test, which was designed to determine the toxicity of the WAF of the test substance, was performed from November 8 to 12, 1996 and was conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test).

The test was performed at 24 ± 1°C under static conditions with a control and five nominal concentrations of test substance (130, 220, 360, 600, and 1,000 mg/L). The WAF was formulated by combining the test substance and dilution water in a glass mixing vessel equipped with a magnetic stirrer, mixing the solution for approximately 20 hours, settling the solution for approximately four hours, and siphoning the water phase containing the water accommodated fraction (WAF). The dilution water was sterile synthetic media adjusted to a pH of 7.5. Nominal concentrations of WAF were used for all calculations.

Algae used for the test (Selenastrum capricornutum, UTEX 1648) was from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin on June 25, 1996. The culture was transferred to sterile enriched media identical to media used for this test and maintained at test conditions for at least 14 days before the definitive test. Algae was incubated under continuous light on a rotary shaker adjusted to 100 rpm. Cell counts were made daily with a haemocytometer.

A determination of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test. After 96 hours of testing, 0.5 ml of solution from each 1,000 mg/L test vessel were combined into one vessel containing 100 ml of fresh media. The resulting solution was incubated under test conditions for 72 hours. During this time, algae increased from a calculated concentration of 12,540 cells/ml to 374,000 cells/ml, indicating that the effect of the test material at the highest tested concentration was algistatic rather than algicidal.

The 96 hour EL50 (presented as “EC50” in report) for both cells per mL and growth rate is >1,000 mg/L nominal WAF loading rate. The 96 hour NOELR (presented as “NOEC” in report) is 360 mg/L nominal WAF loading rate as calculated using the number of cells per mL, and 220 mg/L nominal WAF loading rate as calculated using average specific growth rate.

Test results reported in original study as “effect concentrations (EC)” and “no observed effect concentrations (NOEC)” are reported in this summary as “effect loading (EL)” and “no observed effect loading rate (NOELR)”, respectively, because test results are based on nominal WAF loading rates.

Description of key information

In the key Klimisch 1 study, the acute toxicity of the water accommodated fraction (WAF) was conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test). This resulted in a 96-hour EL50 of > 1,000 mg/L, a NOELR of 220 mg/L, and an EC10 of 840 mg/L for growth rate. The lack of effects is consistent with the supporting study (Ward, 1993), which resulted in a 96-hour EL50 >1,000 mg/L and a NOELR of 1000 mg/L as well as the alkyl phenate sulfide category. 

Key value for chemical safety assessment

EC50 for freshwater algae:

1 000 mg/L

EC10 or NOEC for freshwater algae:

840 mg/L