Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

EC Number:

247-556-2

EC Name:

Dodecylbenzenesulphonic acid, compound with isopropylamine (1:1)

Cas Number:

26264-05-1

Molecular formula:

C18H30O3S.C3H9N

IUPAC Name:

sodium dodecylbenzenesulfonate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control as follows:
Frequency: at 0 and 72 hours
Volume: 2 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the intermediate concentration of nominally 10 mg/L but without algae and samples for analysis were taken at the start and at the end of the test period. Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Test solution preparation:
Preparation of test solutions started with the highest concentration of 100 mg/L applying an overnight period of gentle magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. A layer of foam was observed at the highest test concentration in the combined limit/range-finding test and at the two highest test concentrations in the final test.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms

Test organisms (species):

other: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): Stock and preculture medium M1 (NPR 6505) and M2 (OECD 201 guideline), respectively.
- Any deformed or abnormal cells observed: Not specified

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L in pre-culture medium

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

pH:

The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: 0, 1, 3.2,10 (with and without algae), 32 and 100 mg/L
Measured (initial): 0.026, 0.853, 3.24, 9.05/9.07 (with and without algae), 30.6, and 98.3 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass vessel with aluminium caps
- Type: open
- Material, size, headspace, fill volume: 100 mL containing 50 mL of test solutions
- Aeration: No
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 188.9 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: yes as per OECD 201 guideline

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 83 to 86 µE.m^-2.S^-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the nominal concentration of 32 mg/L and the control to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: As per OECD guidelines
- Range finding study
- Test concentrations: 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: At the end of the test, respectively 8.7 and 40% growth rate inhibition was recorded at nominal concentrations of 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Details on results:

Analysis:
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were at the level of nominal (i.e. 85-101% relative to nominal). During the exposure period, the concentrations remained generally stable, i.e. were at 74-101% of nominal at the end of the test.

Since only the lowest test was slightly below 80% of nominal during at the end of the exposure and, no biologically relevant effect (i.e. >10%) on algal growth was found at this concentration, effect parameters were expressed in terms of analytically confirmed nominal concentrations. It should be noted that small responses were observed in the control samples. Since these responses were caused by carry-over, they were considered irrelevant.

The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae. Hence, it can be stated that the presence of the algae had no effect on the concentration of the test item in test medium throughout the test.

Mean Cell densities:
A concentration-related increase of growth rate and yield inhibition was observed at all concentrations tested, reaching 51% growth rate and 93% yield inhibition at the end of the test. The recorded inhibition of yield and growth rate was statistically significant at all concentrations. However, growth rate inhibition found at the two lowest test concentrations was <10% and therefore considered not biologically relevant. Thus, the NOEC based on biological relevance was set at 3.2 mg/L.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 32 mg/L when compared to the control.

Test Acceptability:
1.     In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 189).
2.     The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 8.3%).
3.     The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.3%).

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 72 hr ErC50 0.9 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L hence the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Determination of NOEC and EC50:
For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).

Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Growth rate and percentage inhibition for the total test period

Rhodacal VS1 Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.746	0.0219	6
1.0	1.696	0.0033	3	2.9^#
3.2	1.655	0.0152	3	5.2^#
10	1.546	0.0389	3	11*
32	1.411	0.0097	3	19*
100	0.863	0.0034	3	51*

* - effect was statistically significant,^#- effect was statistically significant however biologically not relevant (<10%).

Determination of effect concentrations

Parameter (mg/L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<1.0	3.2	12	25	>100
	lower 95%-cl			11	24
	upper 95%-cl			13	27
Yield	Value	<1.0	<1.0	1.0	2.4	12
	lower 95%-cl			0.85	2.1	11
	upper 95%-cl			1.2	2.7	13

cl – confidence limit,* - based on statistical significance,^#- based on biological relevance.

Analytical results:

Time of sampling [hours]	Date of sampling	Date of analysis^1	Concentration [mg/L]			Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed	Corrected^2
0	09 Jul 2018	01 Aug 2018	0	0.031	0.026^4	n.a.
			1	1.02	0.853	85
			3.2	3.88	3.24	101
			10	10.9	9.05	91
			10^3	10.9	9.07	91
			32	36.7	30.6	96
			100	118	98.3	98
72	12 Jul 2018	01 Aug 2018	0	0.056	0.046^4	n.a.	n.a.
			1	0.887	0.740	74	87
			3.2	3.42	2.85	89	88
			10	10.7	8.92	89	99
			10^3	11.3	9.46	95	104
			32	38.6	32.2	101	105
			100	120	100	100	102

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Samples were corrected for the mean accuracy of the QC samples.

³      Without algae.

⁴      Estimated value, calculated by extrapolation of the calibration curve. This is probably caused by carry over since a similar response was observed in the analytical blank.

n.a. Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the test substance inhibited growth rate of the substance although the EC50 could not be determined (> 100 mg/L). The NOEC based on biological relevance was considered to be 3.2 mg/L.