Nitrosylsulphuric acid

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

Study period:

2010-03-15 to 2010-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed and reported in compliance with the OECD Guideline 201, EC and German principles of GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Statement of GLP compliance in report

Test material

Test material information

Constituent 1

Reference substance name:

Nitrosylsulphuric acid

EC Number:

231-964-2

EC Name:

Nitrosylsulphuric acid

Cas Number:

7782-78-7

Molecular formula:

HNO5S

IUPAC Name:

hydroxy(nitrosooxy)sulfane dioxide

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): LZ5593
- Physical state: Viscous liquid
- Purity test date: 2009-11-20
- Expiration date of the lot/batch: 2011-11-20
- Storage condition of test material: Room temperature, protected from moisture and light.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L and control
- Sampling method: The test item contains and partly hydrolyses to sulfuric acid. Thus, the total sulfuric acid content for all concentrations and the
control were analytically verified at the beginning and end (72 h) of the test via UV-spectrophotometry. Separate replicates for the test item analysis at test start were prepared (provided without alga). Samples at the end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis: None, direct analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water. Dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

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:

Not measured

Test temperature:

Mean: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100
pH-Control 7.84 7.11
100 2.94 2.96
50.0 3.52 3.54
25.0 6.22 5.40
12.5 7.04 7.29
6.25 7.29 7.35
Control 7.96 8.12

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1175645 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.

TEST CONCENTRATIONS
- Range finding study: Yes

Results of the Range Finding Test (0-72 h)
Results of the Range Finding Tests (0-72 h)

Nominal
Test Item Concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10 - 4 - 19
1 - 6 - 32
0.1 - 6 - 35
0.01 - 6 - 34
100 (pH adjusted) 21 67

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (31.7 – 34.2)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.614 (CI 0.551 – 0.679) mg/L
Yield Inhibition: 0.333 (CI 0.318 – 0.350) mg/L

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The KOLMOGOROV-SMIRNOV-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. Equal variance test failed for growth rate calculation. The data were transformed using Graph Pad Prism function
Y = Y sqrt.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions
> 10 % are significant. The findings were marked “biologically significant”.

Any other information on results incl. tables

NOEC, LOEC and EC - values of LZ5593(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	25.0
LOEC	50.0
ErC10	26.9 (26.0 – 28.1)
ErC20	29.1 (28.1 – 30.4)
ErC50	33.0 (31.7 – 34.2)
	Inhibition of Yield
NOEC	6.25
LOEC	12.5
EyC10	6.25
EyC20	23.5 (22.2 – 24.7)
EyC50	28.5 (27.2 – 29.8)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
100 pH-Control		1		5074		36734	194752	899866
		2		5074		38854	216962	950809
		3		5074		44098	224748	919583
		Mean		5074		39895	212154	923419
100		1		5074		d.l.	d.l.	3800
		2		5074		d.l.	d.l.	4180
		3		5074		d.l.	d.l.	3800
		Mean		5074		d.l.	d.l.	3927
50.0		1		5074		d.l.	d.l.	4560
		2		5074		d.l.	d.l.	3420
		3		5074		d.l.	d.l.	3420
		Mean		5074		d.l.	d.l.	3800
25.0		1		5074		36591	191034	803512
		2		5074		40191	205175	855045
		3		5074		39887	200574	937545
		Mean		5074		38890	198928	865367
12.5		1		5074		39194	203908	951235
		2		5074		39850	240886	1047409
		3		5074		45111	245633	1085609
		Mean		5074		41385	230142	1028084
6.25		1		5074		37275	206675	1019508
		2		5074		39510	231689	1063891
		3		5074		44180	207352	1116805
		Mean		5074		40322	215239	1066735
Control		1		5074		36935	229684	1390325
		2		5074		38792	232968	1149068
		3		5074		38965	218495	1205538
		4		5074		34754	207573	1018664
		5		5074		40760	238934	1036277
		6		5074		39965	252976	1253996
		Mean		5074		38362	230105	1175645

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100 pH-Control	1		1.73	4.81		894792	23.6
	2		1.74	3.79		945735	19.2
	3		1.73	4.41		914509	21.9
	Mean	(+)^#	1.74	4.34	(+)	918345	21.6
100	1		-0.096	100		-1274	100
	2		-0.065	100		-894	100
	3		-0.096	100		-1274	100
	Mean	(+)	-0.086	100	(+)	-1147	100
50.0	1		-0.036	100		-514	100
	2		-0.131	100		-1654	100
	3		-0.131	100		-1654	100
	Mean	(+)	-0.100	100	(+)	-1274	100
25.0	1		1.69	6.89		798438	31.8
	2		1.71	5.75		849971	27.4
	3		1.74	4.05		932471	20.3
	Mean	(+)^#	1.71	5.56	(+)	860293	26.5
12.5	1		1.75	3.79		946161	19.2
	2		1.78	2.02		1042335	11.0
	3		1.79	1.36		1080535	7.69
	Mean	(-)	1.77	2.39	(-)*	1023010	12.6
6.25	1		1.79	2.51		1014434	13.3
	2		1.78	1.73		1058817	9.55
	3		1.80	0.84		1111731	5.03
	Mean	(-)	1.78	1.69	(-)	1061661	9.31
Control	1		1.87			1385251
	2		1.81			1143994
	3		1.82			1200464
	4		1.77			1013590
	5		1.77			1031203
	6		1.84			1248922
	Mean		1.81			1170571

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.99	1.83	1.80	1.87	0.100	5.32	11.2
	2	2.03	1.79	1.60	1.81	0.220	12.1
	3	2.04	1.72	1.71	1.82	0.186	10.2
	4	1.92	1.79	1.59	1.77	0.168	9.48
	5	2.08	1.77	1.47	1.77	0.308	17.4
	6	2.06	1.85	1.60	1.84	0.232	12.6
				Mean	1.81
				SD ±	0.04
				CV [%]	2.17

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study LZ5593 was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 25.0 and 6.25 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 50.0 and 12.5 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 33.0 (31.7 – 34.2) and 28.5 (27.2 – 29.8) mg/L, respectively.
All effect levels are given based on nominal concentrations of LZ5593.

Executive summary:

The toxicity of LZ5593 to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 from March 15 to 18, 2010. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of LZ5593 were analysed at all concentration levels after 0 and 72 h via UV-spectrophotometry. The recovery rates related to the initial measured concentrations of the component sulphate were calculated to be in the range of 90 to 102 % in the concentration levels of 12.5 mg/L to 100 mg/L (6.25 mg/L: 125 %), demonstrating the stability of the test solutions throughout exposure. All effect values are given based on nominal concentrations of the test item.

NOEC, LOEC and EC - values of LZ5593(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	25.0
LOEC	50.0
ErC10	26.9 (26.0 – 28.1)
ErC20	29.1 (28.1 – 30.4)
ErC50	33.0 (31.7 – 34.2)
	Inhibition of Yield
NOEC	6.25
LOEC	12.5
EyC10	6.25
EyC20	23.5 (22.2 – 24.7)
EyC50	28.5 (27.2 – 29.8)