reaction products of 1 mole of Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs and 1 mole of cyclohexylamine

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Test item solution and control were sampled and immediately analysed when it was possible or stored at -20°C until analysis.

Vehicle:

no

Details on test solutions:

The test solutions were prepared by dilution of a primary stock solution and by directly dissolving the test item in the test solution. There were no difficulty as the test item is a 10% salt dissolved in water.

Test organisms (species):

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

Details on test organisms:

The Pseudokirchneriella subcapitata stock culture was maintained on sloped agar tubes at 4°C and regularly transferred to fresh medium at the laboratory. The algal stock culture was provided by the CCAP (Culture Collection of Algae and Protozoa), reference CCAP 1405/1.
A culture was prepared from the stock culture and maintained in an exponential growth by successive transfers every 3 to 4 days.

Culture conditions are the following:
Medium: OECD medium
Temperature: 23 ± 2°C
Light: continuous illumination between 4440 and 8880 lux

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.1°C - 23.2°C (min-max)

pH:

7.63 - 8.00 (min-max)

Dissolved oxygen:

8.5 - 8.9 mg/L (min-max)

Nominal and measured concentrations:

Purity of test item is 10% (90% of water). Therefore, nominal concentration of active ingredients (cyclohexylamine + dodecylbenzene sulfonate) is one tenth of the nominal concentration of the test item.
Nominal: control ; 10 ; 32 ; 100 ; 320 ; 1000 ; 3200 mg/L test item (i.e., control ; 1 ; 3.2 ; 10 ; 32 ; 100 ; 320 mg/L active ingredients)

Cyclohexylamine and dodecylbenzene sulfonate concentrations were followed independently from each other. For each constituent, concentrations were back calculated into test item concentrations, based on molecular weight, purity and measured concentrations. Each constituent yielded a different test item concentration. The lowest of the two value was considered for ECx determination (worst-case).
Measured: < LoD ; 10.3 ; 28.3 ; 106.1 ; 298.4 ; 1029.1 ; 3216 mg/L test item (i.e.,

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask 250 mL capacity
- Material, size, headspace, fill volume: glass
- Aeration: none
- Type of test: static
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD 201 medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not needed
- Photoperiod: continuous lighting
- Light intensity and quality: 4400 - 8000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

10.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

Results are provided in tables 1 to 4.

Results with reference substance (positive control):

24h-EC50 was 1.34 mg/L

Reported statistics and error estimates:

Growth and yield rates were determined for each concentration. EC10 (Effect Concentration for 10% effect), EC50 and confidence limits were obtained by calculation according to Regtox® Macro (“Vindimian”), using the Hill model. The equation of Hill (1910) was described to model the binding of oxygen to haemoglobin. It was shown to be relevant for many other mechanisms like binding of a ligand to a receptor, Michaelian or non Michaelian enzymatic kinetics, dose or concentration response in toxicology and ecotoxicology (Vindimian 1983, Garric 1990). The model used in this macro is written in the form proposed by Duggleby in 1981. It fits different cases where the measurement of a biological parameter is plotted versus concentration or dose. Two parameters: Hill number and EC50 are characteristics of the probability function. The fitting is based on the algorithm of Marquardt (1963) which is robust and fast. It needs initial estimates of the parameters and optimize them by successive iterations.
The confidence intervals on the parameters are estimated by a bootstrap simulation which is entirely non parametric and is considered to be well adapted to non linear models (Efron 1990). The statistical evaluation (NOEC - No Observed Effect Concentration - and the LOEC - Lowest Observed Effect Concentration - determination) was conducted with R software. Since data were not consistent with a normal distribution (Shapiro-Wilk test) or the variance homogeneity was not confirmed (Bartlett test), data were analysed for significant differences compared to the control using the non-parametric tests Kruskal-Wallis plus Dunn (p = 0.05).

Table 1: Results of the control group

	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)	Specific growth rate 0 h – 24 h	Specific growth rate 24 h – 48 h	Specific growth rate 48 h – 72 h	Growth rate (day^-1)	Yield (cells / mL)
Control	5000	22056	165000	1619000	1,484	2,012	2,284	1,927	1614000
	5000	20344	157000	1378000	1,403	2,043	2,172	1,873	1373000
	5000	22240	203700	2198000	1,492	2,215	2,379	2,029	2193000
	5000	17920	162100	1751000	1,276	2,202	2,380	1,953	1746000
	5000	15984	163900	3899000	1,162	2,328	3,169	2,220	3894000
	5000	14696	202900	1642000	1,078	2,625	2,091	1,931	1637000
Mean	5000	18873	175767	2081167	1,316	2,238	2,412	1,989	2076167

Table 2: Results of the treated groups. Concentrations are given as analytically estimated test item concentration

Rates	Number of cells at 0 h	Measure at 24 h (cells / mL)	Measure at 48 h (cells / mL)	Measure at 72 h (cells / mL)	Growth rate (day^-1)	CV (%)	Growth rate inhibition (%) compared to control	Yield (cells / mL)	CV (%)	Yield inhibition (%) compared to control
mg test item / L
10,269	5000	21320	185400	1348000	1,866	0,347	6,188	1343000	1,948	35,313
	5000	34784	193300	1318000	1,858		6,565	1313000		36,758
	5000	9952	190000	1297000	1,853		6,834	1292000		37,770
Mean	5000	22019	189567	1321000	1,859		6,529	1316000		36,614
28,322	5000	32464	171300	1321000	1,859	0,986	6,527	1316000	5,478	36,614
	5000	18176	173000	1244000	1,839		7,534	1239000		40,323
	5000	10720	180100	1185000	1,823		8,348	1180000		43,164
Mean	5000	20453	174800	1250000	1,840		7,470	1245000		40,034
106,071	5000	11144	112500	959600	1,752	20,109	11,884	954600	65,692	54,021
	5000	7760	114400	1143000	1,811		8,953	1138000		45,187
	5000	8648	49400	198800	1,228		38,270	193800		90,665
Mean	5000	9184	92100	767133	1,597		19,703	762133		63,291
298,364	5000	5760	50280	319300	1,386	1,829	30,328	314300	7,557	84,862
	5000	5848	64480	275100	1,336		32,826	270100		86,990
	5000	8768	67720	299400	1,364		31,407	294400		85,820
Mean	5000	6792	60827	297933	1,362		31,520	292933		85,891
1029,114	5000	6040	31760	37440	0,671	45,808	66,254	32440	86,024	98,438
	5000	2984	16520	12680	0,310		84,402	7680		99,630
	5000	3368	14120	13720	0,336		83,081	8720		99,580
Mean	5000	4131	20800	21280	0,439		77,912	16280		99,216
3215,941	5000	0	14520	9360	0,209	108,166	89,491	4360	120,425	99,790
	5000	0	11760	5720	0,045		97,745	720		99,965
	5000	0	10080	5400	0,026		98,710	400		99,981
Mean	5000	0	12120	6827	0,093		95,315	1827		99,912

Treatment groups significantly different from the control(Kruskal-wallis plus Dunn test, see Appendix).

Table 3: Dissolved oxygen and pH at 0 and 72 h in all treatments

 

	Control	10 mg/L	32 mg/L	100 mg/L	320 mg/L	1000 mg/L	3200 mg/L
pH at 0 h	7.63	7.64	7.75	7.73	7.75	7.83	8
O2at 0 h	8.9
pH at 72 h	7.78	7.93	7.85	7.79	7.70	7.80	7.80
O2at 72 h	8.84	8.7	8.54	8.51	8.58	8.56	8.53

 

Table 4: Conditions of temperature and light intensity throughout the test

Time	0 h	24 h	48 h	72 h
Temperature (°C)	23.1	23.1	22.1	23.2
Light intensity (lux)	8750	8750	8200	8500

 

Validity criteria fulfilled:

yes

Conclusions:

72h-ErC50 and 72h-ErC10 of the active substance "dodecylbenzene sulphonamide compound with cyclohexylamine 1:1" are 47.7 and 10.1 mg/L respectively.

Executive summary:

The objective of this study was to assess the effects of test item (dodecylbenzene sulphonamide compound with cyclohexylamine 1:1) on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201.The test item is a soluble substance under the test conditions and the test solutions were prepared by dilution of a primary stock solution and by directly dissolving the test item in the test solution.

The effects of the test item diluted in OECD test medium at the definitive nominal concentrations of 10; 32; 100; 320; 1000 and 3200 mg/L test item to Pseudokirchneriella subcapitata were investigated under laboratory conditions. Test item quantification was performed according to a validated analytical method using a LC-MS/MS from Sciex (Exion LC and MS QTRAP 5500) as mass spectrometry detection to measure both dodecylbenzene sulphonamide (DDBS) and cyclohexylamine (CHA) from test item concentrations at 0 and 72 hours.Theoretical nominal concentrations values for DDBS and CHA were estimated from their respective molecular weight and purity of the test item (10%). Measured DDBS and CHA concentrations were then back calculated separately from each other in equivalents of test item concentrations.

For both CHA and DDBS, since the deviation from the target or measured initial concentration was not within the range of ± 20 %, statistical analysis of the results were based on the lowest geometric mean exposure concentrations in equivalent of test item (i.e. 10.269, 28.322, 106.071, 298.364, 1029.114 and 2808.751 mg/L test item, or 1.0 ; 2.83 ; 10.6 ; 29.8 ; 102.9 and 280.9 mg/L active substance). 

The study is valid since the growth multiplication factor in the control within the 72-hour test period was higher than 16 (actual value: 416), the coefficient of variation of the average specific growth rates in the control during the whole test period was less than 7% (actual value: 6.2%) and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was less than 35% (actual value: 29.6%).

Based on growth rate:

- The NOEC was 28.3 mg/L test item (i.e., 2.83 mg/L active substance).

- The LOEC was 106.1 mg/L test item (i.e., 10.61 mg/L active substance).

- The 72h-EC50 was 476 mg/L test item (95% confidence limits: 391 – 579 mg.L) (i.e., 47.6 mg/L active substance).

- The 72h-EC10 was 101 mg/L test item (95% confidence limits: 60.3 - 157.9 mg/L) (i.e., 10.1 mg/L active substance).

The concentration of the test item is expressed as the sum of the concentrations of its two moieties (DDBS and CHA).

Description of key information

The objective of this study was to assess the effects of test item (dodecylbenzene sulphonamide compound with cyclohexylamine 1:1) on the growth of Pseudokirchneriella subcapitata over a period of 72 hours according to OECD 201 Guideline. Pseudokirchneriella subcapitata is a freshwater green algae that is recommended by the OECD Guideline 201.The test item is a soluble substance under the test conditions and the test solutions were prepared by dilution of a primary stock solution and by directly dissolving the test item in the test solution.

The effects of the test item diluted in OECD test medium atthe definitive nominal concentrations of 10; 32; 100; 320; 1000 and 3200 mg/L test item to Pseudokirchneriella subcapitata were investigated under laboratory conditions. Test item quantification was performed according to a validated analytical methodusing a LC-MS/MS from Sciex (Exion LC and MS QTRAP 5500) as mass spectrometry detection to measure both dodecylbenzene sulphonamide (DDBS) and cyclohexylamine (CHA) from test item concentrations at 0 and 72 hours.Theoretical nominal concentrations values for DDBS and CHA were estimated from their respective molecular weight and purity of the test item (10%). Measured DDBS and CHA concentrations were then back calculated separately from each other in equivalents of test item concentrations.

For both CHA and DDBS, since the deviation from the target or measured initial concentration was not within the range of ± 20 %, statistical analysis of the results were based on the lowest geometric mean exposure concentrations in equivalent of test item (i.e. 10.269, 28.322, 106.071, 298.364,1029.114 and 2808.751 mg/L test item, or 1.0 ; 2.83 ; 10.6 ;29.8 ; 102.9and 280.9 mg/L active substance). 

The study is valid since the growth multiplication factor in the control within the 72-hour test period was higher than 16 (actual value: 416), the coefficient of variation of the average specific growth rates in the control during the whole test period was less than 7% (actual value: 6.2%) and the mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control was less than 35% (actual value: 29.6%).

Based on growth rate:

- The NOEC was 28.3 mg/L test item (i.e., 2.83 mg/L active substance).

- The LOEC was 106.1 mg/L test item (i.e., 10.61 mg/L active substance).

- The 72h-EC50 was 476 mg/L test item (95% confidence limits: 391 – 579 mg.L) (i.e., 47.6 mg/L active substance).

- The 72h-EC10 was 101 mg/L test item (95% confidence limits: 60.3 - 157.9 mg/L) (i.e., 10.1 mg/L active substance).

The concentration of the test item is expressed as the sum of the concentrations of its two moieties (DDBS and CHA).

Key value for chemical safety assessment

EC50 for freshwater algae:

47.7 mg/L

EC10 or NOEC for freshwater algae:

10.1 mg/L