Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

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

Study period:

From 2004-05-28 to 2004-07-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Concentration of test substance not determined prior to use. Initial concentration of test substance maintained at 74% in the 0.76 mg/l loading rate (instead of 80%). Test solutions prepared individually (dilution of stock solution not appropriate for WAF

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No surrogate or analogue material

Analytical monitoring:

yes

Details on sampling:

Samples of each treatment WAF and the control were taken on Day 0 prior to the addition of algae and at termination (composite of replicates) with no headspace. The samples were stored under refrigeration until analyzed.

Vehicle:

no

Details on test solutions:

Individual treatments were prepared for each loading rate by adding the appropriate amount of test substance to 4.2 L of algal nutrient media and sodium bicarbonate (added as a carbon source in a no headspace environment) in glass aspirator bottles (capacity 4.5 L). The test substance was added to the aspirator bottles using stainless steel and glass syringes. The syringes were weighed with and without the test substance to determine the actual loading rate. The mixing vessels were closed with foil covered rubber stoppers. The mixtures were stirred using a 6.5% (of the static liquid depth) vortex for 24 hours and 10 minutes on magnetic stirplates with Teflon® coated stirbars at room temperature (22.5°C, S.D. 0.4°C). The stirbars were 0.9 cm in diameter and 5.0 cm long, the depth of solution was 23 cm and the mixing speed was approximately 270 rpm. As stirring initiated and after stirring, all treatments appeared clear/colorless with the test substance floating at the surface. The mixtures were allowed to equilibrated to test temperature and the WAFs were removed through the outlet at the bottom of the stirring vessels one hour and five minutes after cessation of stirring.

Test organisms (species):

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

Details on test organisms:

Cultured at the Environmental Toxicology Laboratory of the testing facility. Initial strain (#1648) provided by UTEX, The Culture Collection of Algae MCDB, School of Biological Sciences, The University of Texas at Austin, Austin, TX 78712. Lot # 20 (slant 20B) received by the laboratory on 13-Feb-01.

Culture date: 2004-06-16

Algae are cultured and tested in approximately 300 mL of nutrient media (same as vehicle/dilution water with the exception of additional NaHCO3) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started weekly using inoculum from the previous culture. Cultures of P. subcapitata are
held at 22 - 25°C under continuous illumination (8000 Lux ± 20%) provided by cool-white fluorescent bulbs.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observation period

Test temperature:

Mean test temperature: 23.5°C (sd = 0.2). The temperature of the test solutions at the start of the study was 23.2 to 23.4°C and at termination was 23.3 to 23.5°C.

pH:

7.5-9.8
The pH of each treatment was measured on Day 0 and daily after cell density determinations (composite of the three replicates).

Nominal and measured concentrations:

Nominal loading rates: 0, 0.26, 0.76, 1.7, 4.0 and 10 mg/l
Measured concentrations: see table 2 in §Any other information on materials and mehtods

Details on test conditions:

TEST SYSTEM
Test chambers were 125-mL size autoclaved glass Erlenmeyer flasks sealed with ground glass stoppers to prevent contamination, evaporation and/or volatilization, each containing two 14 mm glass spheres to facilitate mixing. The chambers were filled with approximately 140-mL of the appropriate WAF (no headspace). Test chambers were conditioned with the test solutions prior to the test. The test chambers were placed on shaker tables (100 rpm) to keep the algae in suspension.
- Renewal rate of test solution (frequency/flow rate): No renewal
- Initial cells density:Initial concentration of algae was approximately 1.0 x 104 cells/mL in each replicate chamber.
- No. of vessels per concentration (replicates): 4x3 (3 replicates for each sampling time)
- No. of vessels per control (replicates):4x3 (3 replicates for each sampling time)


GROWTH MEDIUM
See table 1 in § Any information on material and methods

TEST MEDIUM / WATER PARAMETERS
Identical to growth medium

OTHER TEST CONDITIONS
Continuous light: intensity was 8306 to 8426 Lux.
Oscillation Rate: 100 rpm (verified daily).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Cell density was determined for each test and control chamber using a hemacytometer and microscope at 24, 48, 72 and 96 hours (± 1 hour) after the beginning of the test. Cell density determinations were performed on three replicates at each observation interval and the replicates were then discarded.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI not calculated

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (0.48-2.4)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI (1.7-3.0)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.53 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI (0.40-0.75)

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

5.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (3.5-8.6)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (0.92-1.6)

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI not calculated

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI not calculated

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

See tables below

Results with reference substance (positive control):

No reference substance

Reported statistics and error estimates:

The median effect concentration (EbC50) and confidence intervals for inhibition of growth were determined by a probit regression calculation of the probit of the growth inhibition(Ic) vs the log of the concentration and associated confidence intervals based on the methods of Finney. Calculations were based on the PROC PROBIT procedure in SAS.

The NOEC for the EbC50 was based on Duncan’s Multiple Range test7 and Dunnett’s test8 determined from the GLM procedure of SAS6 with percent inhibition (Ic - eqn. 2) as the dependent variable and concentration (c) as the independent variable. The Shapiro-Wilk test9 for normality was used to test if the assumption of normality of the residuals was met; since the residuals were normally distributed the NOEC was based on the estimated values.

The median effect concentration (ErC50) and confidence intervals for growth rate slope were determined by a probit regression calculation of the probit of the growth rate slope (μc – eqn. 3) vs the log of the concentration and associated confidence intervals based on the methods of Finney5. Calculations were based on the PROC PROBIT procedure in SAS6.

The NOEC for the ErC50 was based on Duncan’s Multiple Range test7 and Dunnett’s test8 determined from the GLM procedure of SAS6 with growth rate slope (μc – eqn. 3) as the dependent variable and concentration (c) as the independent variable. The Shapiro-Wilk test9 for normality was used to test if the assumption of normality of the residuals was met; since the residuals were normally distributed the NOEC was based on the estimated values.

Table 3- Percent inhibition

 

Loading rate (mg/l)	Based on the slope of the growth rates		Based on the areas under the growth curves
	72 hours	96 hours	72 hours	96 hours
0.26	-0.5	-0.4	0.7	0.3
0.76	0.1	-0.4	2.1	-0.2
1.7	9.5	5.0	38	33
4.0	61	45	94	93
10	98	90	100	100

Table 4 - Cell concentrations per flask (cells/ml)

 

Loading rate (mg/l)	Day 0	Rep.	Day 1	Rep.	Day 2	Rep.	Day 3	Rep.	Day 4
Control (0)	1.0x10^4	1	5.0 x10^4	4	1.4 x10^5	7	8.4 x10^5	10	2.1 x10^6
	1.0x10^4	2	4.1 x10^4	5	9.4 x10^4	8	9.1 x10^5	11	1.7 x10^6
	1.0x10^4	3	5.0 x10^4	6	9.0 x10^4	9	8.6 x10^5	12	2.1 x10^6
0.26	1.0x10^4	1	3.5 x10^4	4	1.1 x10^5	7	7.9 x10^5	10	2.1 x10^6
	1.0x10^4	2	5.0 x10^4	5	9.5 x10^4	8	9.5 x10^5	11	2.1 x10^6
	1.0x10^4	3	4.8 x10^4	6	1.2 x10^5	9	8.6 x10^5	12	1.7 x10^6
0.76	1.0x10^4	1	4.4 x10^4	4	1.1 x10^5	7	7.9 x10^5	10	1.7 x10^6
	1.0x10^4	2	4.6 x10^4	5	1.1 x10^5	8	7.4 x10^5	11	2.2 x10^6
	1.0x10^4	3	4.5 x10^4	6	1.3 x10^5	9	9.7 x10^5	12	2.2 x10^6
1.7	1.0x10^4	1	3.3 x10^4	4	9.9 x10^4	7	4.5 x10^5	10	1.4 x10^6
	1.0x10^4	2	2.3 x10^4	5	1.2 x10^5	8	5.0 x10^5	11	1.4 x10^6
	1.0x10^4	3	3.4 x10^4	6	9.3 x10^4	9	4.9 x10^5	12	1.7 x10^6
4.0	1.0x10^4	1	1.3 x10^4	4	2.5 x10^4	7	6.1 x10^4	10	2.3 x10^5
	1.0x10^4	2	7.5 x10^3	5	1.6 x10^4	8	3.8 x10^4	11	1.7 x10^5
	1.0x10^4	3	7.5 x10^3	6	4.0 x10^4	9	4.1 x10^4	12	1.4 x10^5
10	1.0x10^4	1	3.8 x10^3	4	8.8 x10^3	7	8.8 x10^3	10	1.4 x10^4
	1.0x10^4	2	5.0 x10^3	5	1.3 x10^4	8	8.8 x10^3	11	1.8 x10^4
	1.0x10^4	3	2.5 x10^3	6	1.0 x10^4	9	6.3 x10^3	12	1.0 x10^4

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EL50 based on growth rate was 4.1 mg/l. The 72-h NOELR based on growth rate was 0.76 mg/l.

Executive summary:

This study was conducted to evaluate the effects of the water-accommodated fractions (WAFs) of Hydrocarbons, C11-C14, isoalkanes, cyclics, aromatics (2-25%) (MRD-04-991) on the growth of the alga, Pseudokirchneriella subcapitata, in a 96-hour static test, conducted according to OECD guideline 201.

Individual treatments were prepared by adding the appropriate amount of test substance to 4.2 L of algal nutrient media and sodium bicarbonate in glass aspirator bottles (capacity 4.5 L) and stirring on magnetic stirplates using an approximately 7% (of the static liquid depth) vortex for approximately 24 hours. After approximately one hour without stirring, the aqueous portions (WAFs) were removed for testing. The actual loading rates were 0 mg/L (control), 0.26, 0.76, 1.7, 4.0, and 10 mg/L. The mean measured concentrations were ND (Not Detected; control), 0.049, 0.16, 0.39, 0.97, and 2.1 mg/L.

Twelve replicate chambers were established for each treatment and the control. The test chambers were completely filled (no headspace) with the appropriate WAF and were sealed with ground glass stoppers. Each chamber contained two 14-mm glass spheres to facilitate mixing. Test chambers were placed on two shaker tables and oscillated at 100 rpm to keep the algae in suspension. The study was performed under continuous light conditions (approximately 8346 Lux) at approximately 24°C. The pH in the test solutions ranged from 7.5-7.6 at the beginning of the test and from 8.1-9.8 at the end of the test. Three replicates from each loading rate were sacrificed daily for cell density determinations.

Acute toxicity results are expressed as the Effect Loading / Effect Concentration 50 (EL/EC50); that is, the loading rate or concentration of test substance in dilution medium which is calculated to result in a 50% reduction in growth derived from either the average specific growth rate (r) or the area under the growth curves (b) relative to the control for the specified time of exposure. The No Observed Effect Loading Rate / No Observed Effect Concentration (NOELR/NOEC) is the highest loading rate or concentration which does not exhibit a statistical difference from the control.

The 72 and 96-hour ErL50 was 4.1 and 5.5 mg/L, respectively. The 72 and 96-hour EbL50 was 2.3 and 2.5 mg/L, respectively. The 72 and 96-hour ErC50 was 0.94 and 1.2 mg/L, respectively.The 72 and 96-hour EbC50 was 0.53 and 0.58 mg/L, respectively. The 72 and 96-hour NOELR was 0.76 mg/L. The 72 and 96-hour NOEC was 0.16 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept. 6, 1996-Dec. 9, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations
- Sample storage conditions before analysis: If samples could not be analysed the day they were taken, they were stored at 4°C and extracted at the earliest opportunity. Samples were stable for up to 7 days.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance was added to measured volumes of test medium. The vessels were then sealed, leaving a small headspace, and the contents stirred with a 1-2 cm vortex depth until the aqueous and test substance phases equilibrated (47.5 hrs). The vessels were then allowed to settle for 1.5-2 hrs and the aqueous phase drawn off for use in the test as a Water Accomodated Fraction (WAF). The control medium was treated in the same manner.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): semi-axenic, Institute of Freshwater Ecology, Windermere
- Age of inoculum (at test initiation): in exponential growth phase
- Method of cultivation: Cultures are made by adding 1.5% (m/v) agar to liquid medium, then autoclaving. Once cooled, it is then poured into 9 cm sterile petri dishes. After setting, the cultures are streaked onto the plates and kept under constant illumination at 21-25 °C in an incubator. The cultures are renewed every two weeks. Liquid medium cultures are initiated from the agar plate cultures with cells transferred on a sterile loop. 100 ml cultures are grown in 250 ml Erlenmeyer flasks for up to 4 days. These cultures are also maintained under constant illumination at 21-25 °C in an incubator.

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.9-23.4 °C

pH:

6.5-9.5

Nominal and measured concentrations:

Nominal loading rate: 0, 0.1, 0.22, 0.46, 1, 2.2, 4.6, 10 mg/l
Measured: <0.05, <0.05, <0.05, 0.10, 0.22, 0.44, 0.79, 1.7 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with glass stoppers
- Material, size, headspace, fill volume: 287 ml Erlenmeyer flask with two teflon marbles added for stirring, placed in a cooled orbital incubator (100 cylces/min)
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 5000 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7 flasks inoculated for controls, 8 flask used for background particle counts

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Made according to Miller, WE, Greene, JC, and Shiroyama, T. (1978). The Selenastrum capricornutum (Prinz) algal assay bottle test. EPA-600/9-78-018. Except 105 µg/l boric acid was added, then 50 mg/l sodium bicarbonate. The medium was autoclaved for at 1.0 kg/cm2 for 15 min, then cooled. The sodium bicarbonate is then added.

OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: 4760 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter or Coulter multisizer every 24 hrs

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

4.6 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.6 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.6 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

4.6 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.6 - 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Growth of Raphidocelis subcapitata at 72 hrs

Concentration	Mean cell Concentration (million cells/ml)	Calculated mean area (A/ 1 million)	Mean reduction in A relative to controls (%)	Mean calculated µ	Mean reduction in µ relative to controls (%)
Control	0.37	6.9	-	0.058	-
0.1	0.36	6.9	0.25	0.058	-0.084
0.22	0.4	7.5	-8.6	0.06	-3.7
0.46	0.34	6.4	7.7	0.057	1.4
1	0.32	6	14	0.056	3.6
2.2	0.29	5.6	19	0.055	4
4.6	0.28	5.2	25	0.054	6.5
10	0.017	0.49	93	0.015	74

Validity criteria fulfilled:

yes

Conclusions:

The test substance exhibited 72-hour EbL50 (biomass) and ErL50 (growth rate) values of 4.6-10 ml/L with the alga, Pseudokirchneriella subcapitata. The 72-hr NOEL values for biomass and growth rate were 0.22 and 1 mg/L, respectively.

Executive summary:

This study was done to determine the acute toxicity of the test material to the alga Pseudokirchneriella subcapitata. The algae were exposed to loading rates of 0, 0.1, 0.22, 0.46, 1.0, 2.2, 4.6, or 10 mg/l (WAF) of test substance for a period of 72 hrs. There was no renewal of test solution. Cell counts were taken at 0, 24, 48, and 72 hrs after start of exposure. The test substance exhibited 72-hour EbL50 (biomass) and ErL50 (growth rate) values of 4.6-10 ml/L with the alga, Pseudokirchneriella subcapitata. The 72-hr NOEL values for biomass and growth rate were 0.22 and 1 mg/L, respectively. 

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept. 6, 1996-Dec. 9, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken during the equilibration phase (WAF preparation: media allowed to settle for about 1 hour) and at the end of the period of use 24 hours later.
All extractions for each test were performed on the day of the samples being taken and all determinations carried out on the same or following day.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Water accomodated fractions of Shellsol H were produced by stirring water appropriate to the test organisms and Shellsol H from 49 hours, leaving the contents of the vessel to stand at least for 1 hour and running off the aqueous phase for use as the test medium.
To enable the Shellsol H to be added at low loading rates, it was added o the growth medium as a solution in acetone and the stirred to prepare the WAF.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Axenic strain (ATCC 22662)
- Source (laboratory, culture collection): American type culture collection, Maryland, Ohio, U.S.A.
- Age of inoculum (at test initiation): in exponential growth phase
- Method of cultivation: Cultures are made by adding 1.5% (m/v) agar to liquid medium, then autoclaving. While cooling, it is then poured into 9 cm sterile petri dishes. After setting, the cultures are streaked onto the plates and kept under constant illumination at 18-22 °C in an incubator. The cultures are renewed every two weeks. Liquid medium cultures are initiated from the agar plate cultures with cells transferred on a sterile loop. 100 ml cultures are grown in 250 ml Erlenmeyer flasks for up to 4 days. These cultures are also maintained under constant illumination at 21-25 °C in an incubator.

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Test temperature:

19-26 °C

pH:

7.4-9.6

Salinity:

Not applicable (freshwater test)

Nominal and measured concentrations:

Nominal loading rate: 0, 0.1, 0.3, 1.0, 3.0, 10, 30 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): sealed
- Material, size, headspace, fill volume: 300 ml Erlenmeyer flask with two teflon marbles added for stirring, placed in a cooled orbital incubator (100 cycles/min)
- initial cell concentration: 5000 cells/ml
- No. of vessels per concentration (replicates): 3 flasks inoculated for control + 1 used for background particle counts
- No. of vessels per vehicle control (replicates): 6 flasks inoculated for controls + 1flask used for background particle counts
- No. of vessels per control (replicates): 3 flasks inoculated for controls + 1 flask used for background particle counts

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water comes from two pumping stations (Highstead and controlled by the Mid Kent Water Company. The water is obtained from bore holes in the chalk of the North Downs. The only chemical treatment prior to its arrival in the laboratory is chlorination to 0.1 mg/l. In the laboratory, the water is filtered (PAL MDY 1001 Y400) to remove all particles larger than 15 µm (90% of particles greater than 10µm) and passed through activated carbon filters (Cuno model CT) to remove chlorine and organic conatminants. Both particle and activated carbon filter cartridges are renewed as recommended by the manufacturers. Heat exchange units (stainless steel and perspex) are used to adjust the temperature of the water.
- Intervals of water quality measurement: six months


OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: 3000 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter counter or Coulter multisizer every 24 hrs

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

10 - 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10 - 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10 - 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

10 - 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

10 - 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

The test substance exhibited 72-hour EbL50 (biomass) and ErL50 (growth rate) values of 10-100 mg/L with the alga, Raphidocelis subcapitata. The 72-hr NOEL values for biomass and growth rate were 3 mg/L, respectively.

Executive summary:

The acute toxicity of water accomodated fractions (aqueous extrats) of Shellsol H has been determined to the unicellular alga Raphidocelis subcapitata (formerly Selenastrum capricornutum).

The test media were prepared by adding quantities of Shellsol H to culture medium appropriate to the test organisms and stirring in sealed vessels for 49 hours). The strirring period was demonstrated as being sufficient to ensure equilibration of the shellsol H and aqueous phases. After stirring the contents of the test vessels were left to settle for about 1 hour before drawing off the WAF for use as the test media in the toxicity tests.

The concentrations of dissolved hydrocarbons (derived from the shellsol H) in the test media was determined during the toxicit tests usig gas chromatography. The analyses showed that the concentration in the test media was <1%% of the loading rate. The reduction in the concentration of dissolved hydrocarbons in the test media during the test was ≤26%.

The acute toxicity to R. subcapitata of water accomodated fractions of Shellsol H prepared at loading ranging from 0.1 to 100 mg/l was determined in a 72h growth inhibition test without renewal of the test media in sealed test vessels. The7h EL50 values (loading rates reducing the area under the growth curves and the average specific growth rates by 50% after 72h exposure) were in the range 10 -100 mg/l.

Description of key information

There is no data available for Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%). However, data is available for structural analogues, Hydrocarbons, C9-C12, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, aromatics (2-25%). This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

Algal cultures were exposed to water accomodated fractions (WAFs) ofHydrocarbons, C9-C12, isoalkanes. cyclics, arornatics (2-25%) in two separate studies.The 72-hr EL₅₀ value for growth rate were found to be 4.1 and 4.6 -10 mg/l. The 72-hr NOELR values for growth rate were 0.76 and 0.22 mg/l, respectively.

The EL₅₀ for algal cultures exposed to WAFs of Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) were 10 -100 mg/l.