Reaction product of di C8-C10, branched, C9 rich, alkylnaphthalene sulphonic acid and alkanolamine

Toxicity to aquatic algae and cyanobacteria

Administrative data

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:

other: GLP study following current guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid: viscous

Details on test material:

Description: Brown highly viscous liquid
Batch: 966018
Purity: UVCB (treated as 100% pure)

Sampling and analysis

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.

Test solutions

Vehicle:

no

Details on test solutions:

A final test was prepared based on the results of a combined limit/range-finding test. Preparation of
test solutions started with individually prepared loading rates between 1.0 and 100 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 off 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. The highest concentration was hazy whereas the remaining concentrations were clear and
colourless with an exception to WAF prepared at 3.2 mg/l which was slightly hazy.
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, 10, 32 and 100 mg/l and solutions
containing 10 and 32% of the WAF prepared at 1.0 mg/l. The initial cell density was 10e+4 cells/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.

Test organisms

Test organisms (species):

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

Details on test organisms:

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

22.8 to 23.5°C.

pH:

7.9 to 8.4

Salinity:

freshwater

Nominal and measured concentrations:

Nominal (WAFs) were prepared at loading rates of 1.0, 3.2, 10, 32 and
100 mg/l and solutions containing 10 and 32% of the WAF prepared at 1.0 mg/l

Measured (mg/L - TWA): 0, 0.015, 0.064, 0.35, 2.41, 0.81, 0.27, 9.6, respectively.

Based on the measured concentrations and the appearance of the solutions at the start of the test,
we have concluded that WAFs prepared at 3.2 and 32 mg/l were exchanged. The measured concentrations
however provide an accurate indication of exposure.

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

Results and discussion

Effect concentrations

Duration:

72 h

Effect conc.:

ca. 0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 0.04 – 0.72 95% CL

Details on results:

Growth rates and yields were in the range of the controls at the three lower TWA concentrations,
whereas the growth rate and yield of algae exposed to higher TWA concentrations were increasingly
reduced, with a maximum growth reduction of 29% and 74% inhibition of yield at the highest
concentration.
Statistically significant reduction of growth rate and inhibition of yield was found at TWA concentrations
of 0.27 mg/l and higher (Bonferroni t-test, α = 0.05), with an exception for the TWA concentration of
0.35 mg/l. For the worst-case-scenario the NOEC was set at 0.064 mg/l.
A floating layer of test substance was observed in WAFs prepared at 3.2, 10 and 100 mg/l. Particles of
test substance were observed in WAFs prepared at 10 and 100 mg/l. In these concentration test
substance adhered to walls of test vessels was observed. Regarding these observations, note that, as
previously discussed, the WAF prepared at 3.2 mg/l was believed inadvertently switched with the WAF
prepared at 32 mg/l. The lack of undissolved material in the 32 mg/l WAF and the presence of test
material particles in the 3.2 mg/l WAF was consisted with this conclusion.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the
exposed cells 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
0.32 mg/l and higher.
The EC50 for growth rate reduction (ERC50: 0-72h) was 0.98 mg/l with a 95% confidence interval
ranging from 0.74 to 1.3 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

Any other information on results incl. tables

TWA conc. Test substance^1 (mg/l)	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.06335		95.00
0.015	0.06418	-1.3	100.83	-6.1
0.064	0.06338	0.0	95.08	-0.1
0.35	0.06227	1.7	88.00	7.4
2.40	0.05373	15.2*	46.92	50.6*
0.81	0.05764	9.0*	63.08	33.6*
0.27	0.05928	6.4*	70.50	25.8*
9.60	0.04529	28.5*	25.17	73.5*

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

* - statistically significant effect (p<0.05).

Parameter	TWA concentration Test substance^1 (mg/l)	95%-confidence interval
NOERC	0.064
72h-ERC10	0.70	0.20 - 2.4
72h-ERC50	>9.6
NOEYC	0.064
72h-EYC10	0.16	0.04 – 0.72
72h-EYC50	2.4	0.53 - 10

Applicant's summary and conclusion

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 and inhibited the yield of this
fresh water algae species significantly at a TWA concentration of 0.27 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 9.6 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 2.4 mg/l with a 95% confidence interval ranging from
0.53 to 10 mg/l.
Statistically seen the LOEC (i.e. Lowest Observed Effect Concentration) was 0.27 mg/l. However,
there was one group with higher TWA concentration but without statistically significant effect. For the
worst-case-scenario the NOEC for both growth rate reduction and yield inhibition was set to
0.064 mg/l. Therefore, in case of this study EC10 provides better indication of toxicity.
The lowest EC10 is for cell number (yield) which is 0.16 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. A final test was prepared based on the results of a combined limit/range-finding test. Preparation of test solutions started with individually prepared loading rates between 1.0 and 100 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 off 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. The highest concentration was hazy whereas the remaining concentrations were clear and colourless with an exception to WAF prepared at 3.2 mg/l which was slightly hazy. 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, 10, 32 and 100 mg/l and solutions containing 10 and 32% of the WAF prepared at 1.0 mg/l. The initial cell density was 10⁴ cells/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 0.038, 0.12, 0.44, 3.5, 1.0, 0.37 and 12 mg/l in 10% of WAF prepared at 1.0 mg/l, 32% of WAF prepared at 1.0 mg/l, WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/l, respectively. The measured concentrations were between 26 and 84% of initial during the test. The decrease of the measured concentration was stronger at the lower concentrations. Based on these results, Time Weight Average concentrations were calculated to be: 0.015, 0.064, 0.35, 2.40, 0.81, 0.27 and 9.60 mg/l, respectively. 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 and inhibited the yield of the fresh water algae species Pseudokirchneriella subcapitata, significantly at a TWA concentration of 0.27 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 9.60 mg/l. The EC50 for yield inhibition (EYC50: 0-72h) was 2.4 mg/l with a 95% confidence interval ranging from 0.53 to 10 mg/l. The NOEC for growth reduction and yield inhibition was 0.064 mg/l. In this study the EC10 provides the best statistical indication of toxicity. The lowest EC10 is for cell number (yield) which is 0.16 mg/L. The EC10 for growth rate is 0.70 mg/L.