Naphthalenesulfonic acid, di-C9-rich C8-10-branched alkyl derivs., calcium salts

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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the
schedule below.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 2 ml
Storage: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

The study was performed based on the results obtained in a previous project 498823 (Toxicity to aquatic algae and cyanobacteria.001). Preparation of test solutions started with individually prepared loading rates between 1.0 and 10 mg/l applying a 2-day period of magnetic stirring. The obtained mixtures were left to settle for a period of 1 hour. Thereafter, the Water Accommodated Fractions (WAFs) were siphoned through glass wool and used as test solutions. The lowest concentrations were prepared by diluting the WAF prepared at a loading rate of 1.0 mg/l in medium. All test solutions were clear and colourless.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

8.0 to 8.2

Salinity:

freshwater

Nominal and measured concentrations:

Nominal (WAFs) were prepared at loading rates of 1.0, 3.2, and 10. Solutions at 2 lower levels were prepared as 10 and 32% dilutions of the WAF prepared at 1.0 mg/l.

At the start of the test, the actual test concentrations were 0.38, 0.79 and 1.8 mg/l for WAFs prepared at 1.0, 3.2 and 10 mg/l. For the 2 lower dilutions, the measured values were 0.039 and 0.12 mg/l for 10 and 32% of the WAF prepared at 1.0 mg/l.

Details are presented in the table section.

Details on test conditions:

Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 104 cells/ml.
Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30
Watt, with a light intensity within the range of 68 to 77 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator
and as such were daily repositioned. During incubation the
algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 0.061 – 0.63 95% CL

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.29 - 2.2

Details on results:

Growth rates were in the range of the controls at all concentrations besides the highest one 72-hour test period. In the highest concentration tested 13% reduction of growth was observed. Although statistically significant reduction of growth rate was found at an average exposure concentrations of 0.30 mg/l and higher (Bonferroni t-test, α = 0.05), the NOEC was set to 0.57 mg/l. This was done because the effects observed at 0.30 and 0.57 mg/l were biologically not relevant (i.e. <10%).

Inhibition of yield increased with increasing concentration of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) from the lowest concentration tested, resulting in 50% inhibition at the average exposure concentration of 1.2 mg/l. Statistically significant inhibition of yield was found at test concentrations of 0.019 mg/l and higher (Bonferroni t-test, α = 0.05).
EC10 values are considered the most valid estimate for risk assessment as they take into consideration the full dose - response pattern of the test. The EC10 for yield (cell number) is 0.20 mg/L and 0.80 mg/L for growth rate.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the cells exposed to the highest concentration when compared to the control.

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations
0.18 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.97 mg/l with a 95% confidence interval
ranging from 0.62 to 1.5 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3
mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (EYC50: 0-72h) was 0.44 mg/l with a 95% confidence interval ranging from
0.32 to 0.61 mg/l. The historical ranges of the 72h EC50 for yield inhibition lie between 0.43 and 1.1
mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

see tables

Measured concentrations versus nominal concentrations

Test substance^1 Loading rate (mg/l)	Measured cocnentrations (mg/l)			Average exposure concentration (mg/l)
	t=0h	t=24h^2	t=72h^2
10% of 1.0	0.0393	0.0084^3	0.00809	0.019
32% of 1.0	0.124	0.0520	0.0569	0.078
1.0	0.383	0.282	0.236	0.30
3.2	0.789	0.398	0.529	0.57
10	1.75	0.626	1.31	1.23
10 wa	1.71	0.620	1.33	1.22

1. di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

wa – without algae

2. Arithmetic mean of two measurements

3. Based on only one run as one sample broke during storage.

Test substance^1 Average exposure concentration (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.07597		236.78
0.019	0.07389	2.7	203.71	14.0*
0.078	0.07347	3.3	197.48	16.6*
0.30	0.07294	4.0^(*^)	190.19	19.7*
0.57	0.07025	7.5^(*^)	158.81	32.9*
1.2	0.06632	12.7*	117.95	50.2*

1. di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA)

* - statistically significant effect (p<0.05)

^(*)– Effect statically significant but biologically irrelevant (<10%).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) reduced growth rate of this fresh water algae species significantly at an average exposure concentration of 1.2 mg/l.

The EC50 for growth rate reduction (72h-ERC50) was beyond the range tested, i.e. exceeded an average exposure concentration of 1.2 mg/l.

The EC50 for yield inhibition (72h-EYC50) was 1.2 mg/l with a 95% confidence interval ranging from 0.41 to 3.5 mg/l.
The EC10 for growth inhibition was 0.80 mg/l with a 95% confidence interval ranging from 0.29 to 2.2 mg/l.

The EC10 value for yield inhibition was 0.20 mg/l with a 95% confidence interval ranging from 0.061 to 0.63 mg/l.

Executive summary:

A Fresh Water Algal Growth Inhibition Test was conducted for di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) with Pseudokirchneriella subcapitata. The study procedures were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. The batch of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) tested was a brown highly viscous liquid and a UVCB substance (treated as 100% pure). The test substance was not completely soluble in test medium at the concentrations tested.

The study was performed based on the results obtained in a previous project 498823 (Toxicity to aquatic algae and cyanobacteria.001). Preparation of test solutions started with individually prepared loading rates between 1.0 and 10 mg/l applying a 2-day period of magnetic stirring. The obtained mixtures were left to settle for a period of 1 hour. Thereafter, the Water Accommodated Fractions (WAFs) were siphoned through glass wool and used as test solutions. The lowest concentrations were prepared by diluting the WAF prepared at a loading rate of 1.0 mg/l in medium. All test solutions were clear and colourless.

 

Six exponentially growing algal cultures were exposure to an untreated control whereas three replicates per group were exposed to WAFs prepared at 1.0, 3.2 and 10 mg/l and solutions containing 10 and 32% of the WAF prepared at 1.0 mg/l. The initial cell density was 10⁴cells/ml. The total exposure time was 72 hours and samples for analytical confirmation of the exposure concentrations were taken at times 0, 24 and 72 hour of exposure.

 

At the start of the test, the actual test concentrations were 0.38, 0.79 and 1.8 mg/l for WAFs prepared at 1.0, 3.2 and 10 mg/l and 0.039, 0.12 mg/l for 10 and 32% of the WAF prepared at 1.0 mg/l.

The measured concentrations were between 21 and 75% of initial after 24 hours of exposure and between 17 and 76% of initial at the end of the test. The calculated average exposure concentrations corresponded to 0.019, 0.078, 0.30, 0.57 and 1.2 mg/l for 10, 32 and 100% of WAF prepared at 1.0 mg/l and WAFs prepared at 3.2 and 10 mg/l.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid (DNNSA) reduced growth rate of this fresh water algae species significantly at an average exposure concentration of 1.2 mg/l.

 

The EC₅₀for growth rate reduction (72h-E_RC₅₀) was beyond the range tested, i.e. exceeded an average exposure concentration of 1.2 mg/l.

 

The EC₅₀for yield inhibition (72h-E_YC₅₀) was 1.2 mg/l with a 95% confidence interval ranging from 0.41 to 3.5 mg/l.

 

The EC₁₀ for growth inhibition was 0.80 mg/l with a 95% confidence interval ranging from 0.29 to 2.2 mg/l.

 

The EC₁₀value for yield inhibition was 0.20 mg/l with a 95% confidence interval ranging from 0.061 to 0.61 mg/l.

Description of key information

The toxicity to aquatic algae for CALCIUM BIS( DI C8-C10, BRANCHED, C9 RICH, ALKYLNAPHTHALENE SULPHONATE) is determined by read-cross from testing with the close structural analog DNNSA (di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid). The EC50 value in the DNNSA algae test for growth rate is >1.2 mg/L. The EC10 for growth rate is 0.8

Based on these findings no toxicity is expected at the water solubility level of CaDNNSA.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.2 mg/L

EC10 or NOEC for freshwater algae:

0.8 mg/L

Additional information

Under the conditions of the DNNSA study with Pseudokirchneriella subcapitata, reduced growth rate of this fresh water algae species significantly at a TWA concentration of 0.30 mg/l and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was beyond the range tested, i.e. exceeded a TWA concentration of 1.2 mg/l. The lowest EC10 in this study is for cell number (yield) which is 0.2 mg/L and the EC10 for growth rate is 0.80 mg/L. This last value is the key value for safety assessment.