4-poly(propyl), benzene sulfonic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

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

Adequacy of study:

key study

Study period:

11 October 2007 to 6 February 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: AS305BD
Description: dark brown, viscous liquid
Batch number: TS07002
Analytical purity: 100%
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

A Strata C8 (500 mg/3 mL) solid phase extraction (SPE) cartridge containing glasswool was sequentially preconditioned with methanol and water. A volume (300 mL) of test sample was eluted through the cartridge and the cartridge dried on the SPE manifold. The test material was eluted from the cartridge with methanol (5 mL).

Samples were taken at the start and end of the range-finding test and at 0, 23, 47, 72 and 96 hours in the definitive test.

Test solutions

Vehicle:

no

Details on test solutions:

An amount of test material (1222 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by filtration through a 0.2 um Sartorius Sartopore filter (first approximate 1 litre discarded in order to precondition the filter) to give a saturated solution with a concentration of 3.25 mg/L as AS305BD. A series of dilutions was made from this saturated solution to give further stock solutions of 2.88, 2.11, 1.18 and 0.786 mg/L. An aliquot (1 litre) of each of the stock solutions was separately inoculated with algal suspension (9 mL) to give the required test concentrations of 0.786, 1.18, 2.11, 2.88 and 3.25 mg/L as AS305BD.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa ((CAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland

ACCLIMATION
- Culturing media and conditions (same as test or not): yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not stated

Test temperature:

24 ± 1°C

pH:

7.5±0.1

Dissolved oxygen:

not reported.

Salinity:

Not applicable.

Nominal and measured concentrations:

Based on the results of the range-finding test, test material solutions for the definitive test were prepared by stirring an excess (100 mg/L) of test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a 100% v/v saturated solution. This saturated solution was then further diluted, as necessary, to produce the remaining test groups of 50, 25, 12.5 and 6.25% v/v saturated solution.

Analysis of the test solutions at 0 hours showed measured concentrations of 0.786, 1.18, 2.11, 2.88 and 3.25mg/L as AS305BD.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks
- Initial cells density: 4 x 10E+3 cells/ml
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: 7000 lux

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H20 4.41 mg/L
MgS04.7H20 14.7 mg/L
K2HP04 1.044 mg/L
NaIHCO3 15.0 mg/L
H3B03 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCI3.6H2O 0.159 mg/L
CoCh2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H20 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified deionised water* and the pH
adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCL.
*Elga Optima 15+ or Elga Purelab Option R-15 BP.

- Culture medium different from test medium: no
- Intervals of water quality measurement: 24 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
Range finding test: Samples were taken at the start and end of the test and measured with a Coulter® Multisizer Particle Counter.
Definitive test: Samples were taken at 0, 23, 47, 72 and 96 hours and the cell densities detennined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0, 0.786, 1.18, 2.11, 2.88, 3.25 mg/l (measured).

- Range finding study
- Test concentrations: 1.0, 10, 100 % v/v saturated solution (nominal)
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

3.12 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.88 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Effect concentrations 3

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3.25 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

growth rate

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.96 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: yield

Effect concentrations 5

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.88 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: yield

Effect concentrations 6

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3.25 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

other: yield

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

2.99 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Effect concentrations 8

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

2.88 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Effect concentrations 9

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

3.25 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Effect concentrations 10

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.78 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 11

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 12

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

1.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 13

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.73 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 14

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 15

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

1.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 16

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 17

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 18

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

1.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

See attached Tables 1 to 5

INHIBITION OF GROWTH RATE
Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 0.786, 1.18, 2.11 and 2.88 mg/L test concentrations (P<0.05). However, the test concentration of 3.25 mg/L was significantly different (P<0.05) and therefore the NOEC based on growth rate was 2.88 mg/L. Correspondingly the LOEC based on growth rate was 3.25 mg/L.

INHIBITION OF YIELD
Statistical analysis of the yield data at 72 and 96 hours was carried out as detailed above. There were no statistically significant differences between the control, 0.786, 1.18, 2.11 and 2.88 mg/L test concentrations (p <0.05). However, the test concentration of 3.25 mg/L was significantly different (P<0.05) and therefore the NOEC based on yield was 2.88 mg/L. Correspondingly the LOEC based on yield was 3.25 mg/L.

INHIBITION OF BIOMASS INTEGRAL
Statistical analysis of the biomass integral data at 72 and 96 hours was carried out as detailed above. There were no statistically significant differences between the control, 0.786, 1.18, 2.11 and 2.88 mg/L test concentrations (P< 0.05). However, the test concentration of 3.25 mg/L was significantly different (P

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after
96 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Any other information on results incl. tables

RANGE-FINDING TEST

The results showed no effect on growth at the test concentrations of 1.0 and 10% v/v. However, growth was observed to be reduced at 100% v/v. Based on this information the test material solutions for the defmitive test were prepared by stirring an excess (l00 mg/L) of test material in culture medium for a period of time and then removing any undissolved test material by filtration to give a 100% v/v saturated solution. This saturated solution was then further diluted, as necessary, to produce the remaining test groups of 50, 25, 12.5 and 6.25% v/v saturated solution. Analysis of the test solutions at 0 hours showed measured concentrations of 0.786, 1.18, 2.11, 2.88 and 3.25 mg/L as AS305BD.

VERIFICATION OF TEST CONCENTRATIONS

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.786 to 3.25 mg/L as AS30SBD. analysis of the test preparations at 96 hours showed a decline in measured test concentrations in the range of 0.177 to 1.02 mg/L as AS305BD. This decline, in excess of that seen in the stability analyses was considered to be due to possible adsorption of the test material to the algal cells present. Whilst no immediate adsorption was observed in the recovery analyses conducted in the presence of algal cells this does not preclude long-term adsorption over the test period. Adsorption was not a factor in the stability analyses as no algal cells were present. Current regulatory advice is that, in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The geometric mean measured test concentrations were determined to be:

0-hour measured test concentration (mg/L)	Geometric mean measured test concentration (mg/L)
0.786	0.37
1.180	0.69
2.110	0.99
2.880	1.71
3.250	1.82

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

validity criteria (as above) fulfilled.

Conclusions:

The 96 h ErC50, EyC50 and EbC50 were determined to be 3.12 mg/L, 2.96 mg/L and 2.99 mg/L. Based on geometric mean test concentrations these values are 1.78 mg/L, 1.73 mg/L and 1.74 mg/L.

The 96-h NOEC and LOEC based on growth rate, yield and biomass integral were 2.88 mg/L and 3.25 mg/L respectively. Based on geometric mean measured test concentrations these values are 1.71 mg/L and 1.81 mg/L.

Executive summary:

A study was performed to assess the effect of the test material on the growth of alga in line with OCED Guideline 201. P. subcapitata was exposed to test concentrations of 6.25, 12.5, 25, 50 and 100% v/v equivalent to 0.786, 1.18, 2.11, 2.88 and 3.25 mg/L (three replicates per concentration).

The following results were based on nominal concentrations:

Time Point	Response variable	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
72 h	Growth rate	3.22	2.88	3.25
	Yield	2.99	2.88	3.25
	Biomass integral	3.05	2.88	3.25
96 h	Growth rate	3.12	2.88	3.25
	Yield	2.96	2.88	3.25
	Biomass integral	2.99	2.88	3.25

Analysis of the test preparations showed a decline in measured test concentrations. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a 'worst-case' analysis of the data:

Time Point	Response variable	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
72 h	Growth rate	1.81	1.71	1.82
	Yield	1.74	1.71	1.82
	Biomass integral	1.76	1.71	1.82
96 h	Growth rate	1.78	1.71	1.82
	Yield	1.73	1.71	1.82
	Biomass integral	1.74	1.71	1.82