Hydrocarbons, C11-C13 (odd numbered), n-alkanes, <2% aromatics

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance is a mixture that is poorly soluble in water (information from sponsor). Therefore a water accommodated fraction (WAF) was prepared using a slow-stir liquid-liquid saturator technique. Two glass aspirator bottle (2-L) with a bottom side-outlet attached to a stopcock were filled with 2-L test media. The necessary volume of the test substance was calculated from the density. Then 267-µL (100 mg/L considering density = 0.75 g/cm³) per bottle of the test substance was pipetted carefully on the water surface. The bottles were closed and the solutions were stirred on a magnetic plate for approximately 1 day. The stirring was slow (approx. 130 rpm) so that no vortex formed. The undissolved test substance remained on the surface and the required volume of the WAF (approx. 1.5-L) was removed from the bottom of the bottles and again visually inspected for the presence of any undissolved test substance. The saturated solutions from both bottles were combined and used for testing.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No undissolved test substance was observed in the merged test solution. The test solution was visibly clear following preparation.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: SAG 61.81
- Source (laboratory, culture collection): collection of algal cultures in Göttingen, Germany
- Method of cultivation: A stock algal culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared from the stock culture and incubated for 4 days at 21 – 24 °C (max. temperature difference 2 °C). After this time, the inoculum culture is in exponential growth phase and can be used to initiate the test (study day 0).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 °C

pH:

7.4 -7.5 (at test start)
7.4 - 10.0 (at test end)

Nominal and measured concentrations:

Nominal: 100 mg/L as loading rate WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (nominal volume 250 mL) containing 100 mL test solution plugged with gas permeable silicone sponge caps
- Type (delete if not applicable): closed
- Initial cells density: 0.3E4 cells/mL
- Control end cells density: 321E4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- other: continuous stirring

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Alkalinity: To accommodate testing in a closed system (see Mayer 2000; ISO/DIS 14442, 2004) the concentration of NaHCO3 in the final modified OECD medium was increased to 300 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH at start and at end of test, temperature continuously

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: permanent illumination
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25). To minimize the potential effect of slight variations in illumination, the test vessels were rearranged daily. Average 5666 lux (within ± 15% variability) at a wave length of 400 - 700 nm. Light intensity was adjusted to the lower end of the acceptable range, to control pH changes due to excessive algal growth in the closed test vessels.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: After the end of the exposure the control replicates were mixed and serially diluted by factor 2. The fluorescence of aliquots from the undiluted mixture and the dilutions were measured and in parallel cell density was determined by a direct microscopic count (two counts in a Neubauer haemocytometer). These data were used to derive a linear correlation between fluorescence and cell density.
- Chlorophyll measurement: Algal growth measured as in vivo chlorophyll-a fluorescence (pulsed excitation with light flashes having a wavelength of 430 nm).
- other: The inoculum culture was observed microscopically at the start of the test to verify normal and healthy cells. At test termination, a pooled sample from each test concentration was examined and any abnormal appearance of the algae noted.

TEST CONCENTRATIONS
- Range finding study: yes
- Results used to determine the conditions for the definitive study: In the preliminary range finding test a concentration of 100 mg/L caused no inhibition of algal growth relative to the control after 72 hours. According to the OECD-guideline, the highest suggested test concentration is 100 mg/L for a limit test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(WAF)

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: the treatment show a slightly higher growth rate (+4.8%) compared to the control.

Results with reference substance (positive control):

The ErC50 (72 h) of the control substance potassium dichromate was 1.05 mg/L (Date of the last control experiment: 13 Feb 2015 , project number: 60E0063/04E006). These results indicate that the algae are responding normally to toxicant stress.
An additional reference toxicant test was conducted with the same method modifications described in this study (modified exposure media, closed test system, and reduced inoculation density) to determine any effect on algal response to toxic stress. The ErC50 (72 h) of the control substance potassium dichromate was 1.32 mg/L (Date of experiment: 23 Feb 2015 , project number: (60E0063/04E007). These results indicate that the algae are responding normally to toxicant stress under the modified test conditions and that these deviations from test guidelines do not affect the results of this study.

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The water accommodated fraction of the test material were prepared by stirring the test material in the exposure solution for 25 hours after which the the aqueous phase was removed for testing.

Test organisms (species):

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

Details on test organisms:

Organisms used in the test were from a laboratory culture, originally derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, Ohio, USA.

Test type:

static

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

24 to 26 degrees C

pH:

Within Guideline requirements

Nominal and measured concentrations:

The nominal loading rate was 1000 mg/L. A control was also tested.

Details on test conditions:

The study was conducted in sealed test systems with no headspace that were not renewed during the study. The test systems used were 300 ml glass Erlenmeyer flasks. The treatment level was evaluated in triplicate test systems and the control in 6 replicate test systems. Control and treatment systems were innoculated with a concentration of 5000 cells/ml. Test systems were incubated under constant illumination (approximately 3000 lux) on an orbital incubator.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

The growth of algae in the the test systems was greater than the controls, which performed normally, therefore no analyses were required.

Validity criteria fulfilled:

yes

Conclusions:

Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.

Executive summary:

Growth of alga cultures, as measured by biomass and growth rate, exposed to a water accommodated fraction of the test substance was not inhibited over 72 hours. The growth of algae in the the test systems was greater than the controls, which performed normally. Therefore, the 72-hr EL50 values for the two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are reported as 1000 mg/L, respectively.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

March to November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

An equilibration study was conducted prior to the study to determine the length of time needed to develop water accommodated fractions (WAFs) at the 1000 mg/L loading level. The aqueous phase of the WAF was analyzed after 1, 24, 48, and 96 hours of stirring. In all samples, the quantity of test substance in solution was less than the limit of detection (0.5 mg/L). Further analysis of test substance in test media a loading level of 1 g/L also showed no detectable amounts of test substance. Consequently, analysis of treatment solutions from the definitive study were not conducted.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

Organisms used in the test were from a laboratory culture, originally derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, Ohio, USA.

Test type:

static

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

23 to 25 degrees C

pH:

7.2 to 8.8

Nominal and measured concentrations:

The nominal loading rate was 1000 mg/L. A control was also tested.

Details on test conditions:

The study was conducted in sealed test systems with no headspace that were not renewed during the study. The test systems used were 300 ml glass Erlenmeyer flasks that were complely filled with test media. Six replicate test systems were established for the treatment level and control. Control and treatment systems were innoculated with a concentration of 5000 cells/ml. Test systems were incubated under constant illumination (approximately 3000 lux) on an orbital incubator.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

None

Validity criteria fulfilled:

yes

Conclusions:

Growth in an alga culture, as measured by biomass and growth rate, exposed to a 1000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >1000 mg/L and 1000 mg/L, respectively.

Executive summary:

Growth in an alga culture, as measured by biomass and growth rate, exposed to a 1000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >1000 mg/L and 1000 mg/L, respectively.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

September 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Skeletonema costatum

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20.3 to 20.9 degrees C

pH:

7.5 to 9.2

Nominal and measured concentrations:

Nominal test concentrations were 10094, 20171, 30135, 50069, 60021, 80072, and 100137 mg/L. A control was also tested.

Details on test conditions:

Water accommodated fractions of the test material were prepared for each loading level.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

13 906 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 12343 to 15406

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 60 021 - < 80 072 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI not determined

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

57 161 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 54701 to 59867

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 137 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI not determined

Duration:

72 h

Dose descriptor:

EL90

Effect conc.:

234 959 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI = 207059 to 272344

Duration:

72 h

Dose descriptor:

EL90

Effect conc.:

> 100 137 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI not determined

Results with reference substance (positive control):

The growth inhibition of Skeletonema costatum was evaluated with 3,5-dichlorophenol as a positive control at a concentration of 1.20 mg/L, which produced 56% growth inhibition.

Validity criteria fulfilled:

yes

Conclusions:

Alga (Skeletonema costatum) growth, as measured by biomass, exposed to water accommodated fractions of the test substance exhibited EL10, EL50, and EL90 values of 13906, 57161, and 234959 mg/L, respectively. In comparison, the growth rate results for the endpoints EL10, EL50, and EL90 were 60021 to 80072, >100137, and >100137 mg/L, respectively.

Executive summary:

Alga (Skeletonema costatum) growth, as measured by biomass, exposed to water accommodated fractions of the test substance exhibited EL10, EL50, and EL90 values of 13906, 57161, and 234959 mg/L, respectively. In comparison, the growth rate results for the endpoints EL10, EL50, and EL90 were 60021 to 80072, >100137, and >100137 mg/L, respectively.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

February 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.

Qualifier:

according to guideline

Guideline:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Skeletonema costatum

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

20.3 to 21.5 degrees C

pH:

9.2 to 9.4

Nominal and measured concentrations:

Nominal loading rates were 10038, 20012, 30052, 60195, and 100002 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Results with reference substance (positive control):

The growth inhibition of Skeletonema costatum was evaluated with 3,5-dichlorophenol as a positive control at a concentration of 1.21 mg/L, which produced 79% growth inhibition.

Validity criteria fulfilled:

yes

Conclusions:

Alga (Skeletonema costatum) growth, as measured by biomass and growth rate, in cultures exposed to a 100,000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >100,000 mg/L and 100,000 mg/L, respectively.

Executive summary:

Alga (Skeletonema costatum) growth, as measured by biomass and growth rate, in cultures exposed to a 100,000 mg/L water accommodated fraction of the test substance was not inhibited when compared to the control after a 72-hour exposure. Therefore, the 72-hr EL50 and NOELR values for these two endpoints are reported as >100,000 mg/L and 100,000 mg/L, respectively.

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

January to May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This robust summary has a reliability rating of 1 because the study followed a standard guideline, followed GLP guidelines, and was conducted without deviations that would invalidate the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

Organisms used in the test were from a laboratory culture, originally derived from a strain (ATCC 22662) obtained from the American Type Culture Collection, Maryland, Ohio, USA.

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test temperature:

23.8 to 24.4 degrees C

pH:

Within Guideline requirements

Nominal and measured concentrations:

The nominal loading rates were 1, 3, 10, 30, 100, 300, and 1000 mg/L. A control was also tested.

Details on test conditions:

The study was conducted in sealed test systems with no headspace that were not renewed during the study. The test systems used were glass Erlenmeyer flasks that contained 287 ml when full. Each treatment level was evaluated in triplicate test systems and the control in 6 replicate test systems. Control and treatment systems were innoculated with a concentration of 5000 cells/ml. Test systems were incubated under constant illumination (approximately 5000 lux) on an orbital incubator.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Reported statistics and error estimates:

Statistical analysis of the EL50 data were not conducted becasue a 50% effect was not achieved. A William's test (William, 1971, 1972) was used to calculate the significance of difference in area under the growth curve and average specific growth rate data from the control and treatment systems to determine the NOELR values (calculated at a 5% probability level).

Validity criteria fulfilled:

yes

Conclusions:

Growth of alga cultures, as measured by biomass and growth rate, exposed to water accommodated fractions of the test substance was not inhibited by more than 50% over 72 hours. Therefore, the 72-hr EL50 values for these two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are 3 and 100 mg/L, respectively.

Executive summary:

Growth of alga cultures, as measured by biomass and growth rate, exposed to water accommodated fractions of the test substance was not inhibited by more than 50% over 72 hours. Therefore, the 72-hr EL50 values for these two endpoints are reported as >1000 mg/L. The 72-hr NOELR values for biomass and growth rate are 3 and 100 mg/L, respectively.

Description of key information

EL50 (72h) > 100 mg/L and NOELR (72h) >= 100 mg/L (nominal, WAF) for growth rate of Pseudokirchneriella subcapitata (OECD 201)

Additional information

One study investigating the toxicity of Hydrocarbons, C11-C13 (odd numbered), n-alkanes, < 2% aromatics to freshwater algae is available. The study was performed according to OECD guideline 201 under GLP conditions (BASF 2015). A 100 mg/L loading rate WAF was tested in a limit test. No growth rate inhibition was observed in the treatment compared to the control. Thus, the 72h-EL50 value is determined to be > 100 mg/L and the 72h-NOELr value is determined to be >= 100 mg/L based on the nominal concentration. The very low acute toxicity is confirmed by experimental data provided for very similar hydrocarbons in the range of C9 to C14.