2-hydroxy-4-(methylthio)butyronitrile

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:

From 11/06/2010 (study plan) to 26/08/2010 (date final report)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed in accordance with OECD TG and GLP. Due to the degradation properties of the test item and in accordance with the OECD guidance document on difficult substances (OECD SERIES ON TESTING AND ASSESSMENT Number 23), the results obtained take into account the toxicity of the degradation products (see discussion in the field "overall remarks"). But due to the uncertainties on the concentrations of the parent and degradation products tested, this study is considered as reliable with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-4-(methylthio)butyronitrile

EC Number:

241-756-3

EC Name:

2-hydroxy-4-(methylthio)butyronitrile

Cas Number:

17773-41-0

Molecular formula:

C5H9NOS

IUPAC Name:

2-hydroxy-4-(methylsulfanyl)butanenitrile

Details on test material:

See "confidential details on test material"

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae). Concentrations: 0.1, 0.32, 1, 3.2 and 10 mg/L. At the same sampling time, duplicate samples were also taken from the control.
- Sampling method: No additional data.
- Sample storage conditions before analysis: Aliquots of 10 mL of the samples were diluted with 2.5 mL methanol and 6.25 µL phosphoric acid giving a sample preparation factor of 1.25. The final solutions were measured directly without storage.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium of the highest nominal concentration of 100 mg/L was prepared by dissolving 29.94 mg of the test item completely in 3000 mL of test water using intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at Harlan Laboratories under standardized conditions according to the test guidelines.

An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

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:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C.

pH:

The pH was measured and recorded in each treatment at the start and at the end of the test. It was found to be 8 in all treatments at the start of the test, and to vary from 8.1 to 9.2 at the end of the test.

Dissolved oxygen:

Not measured.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal concentrations were 10; 3.2; 1; 0.32 and 0.10 mg/L.
The initialy measured concentrations were respectively : 5.6 mg/L (56% of nominal); 1.4 mg/L (42% of nominal); 0.40 mg/L (40% of nominal); 0.14 mg/L (45% of nominal) and 0.033 mg/L (for this concentration the average concentration was < LOQ (Limit of quantification, LOQbio: 0.0668 mg/L); the 1/2 LOQbio (0.0334 mg/L) was used as start value and for calculation of the EC-values).

Details on test conditions:

TEST SYSTEM
- Test vessel:
50 mL Erlenmeyer flasks (glass) were used per replicate containing 15 mL of test solution. Each test flask was covered with a glass dish. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density:
The test was started using a nominal algal cell density of 10000 cells/mL. The initial cell density was selected according to the recommendations of the OECD test guideline. The algal cell density in the pre-culture was determined by an electronic particle counter (Coulter Counter, Model ZM).
- Control end cells density: mean of 287000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water to obtain the adequat concentrations.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: constant illumination
- Light intensity and quality: The mean measured light intensity at the level of the test solutions was approximately 7600 Lux (range: 6670 to 8350 Lux, measured at nine places in the experimental area). The light intensity over the incubation area (measured at nine places in the experimental area) was within ±15% from the average light intensity as recommended by the guideline.

EFFECT PARAMETERS MEASURED :
A small volume of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.
The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the test concentration of nominal 1 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected. This test concentration was chosen since the algal cell density was too low for a reliable examination at the other highest nominal concentrations.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: The enlarged spacing factor of 3.2 between the test concentrations was chosen based on the results of the range-finding test, which documented a rather flat concentration-effect relationship. Therefore, a large concentration range had to be tested.
- Range finding study: The selection of the test concentrations was based on the results of a range-finding test (non GLP), but no details are reported in the test report.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control : yes

- Observation of abnormalities / Unusual cell shape : The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 1.0 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

- Observation of the test media: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

- Stability of the test item:
The measured concentrations of the test item in the test media of the nominal test concentrations of 0.32 to 10 mg/L were between 40 and 56% of the nominal values at the start of the test. The measured concentration of the test item in the test medium of the nominal test concentration of 0.10 mg/L was below LOQ (Limit of quantification, LOQbio: 0.0668 mg/L, see Table 4 in the field "any other information on results including tables").
During non-GLP pre-experiments on stability of the test item it has been observed that under the conditions of the algal toxicity test the test item degrades into degradation products (see also the field "Overall remarks"). As the degradation products are known to inhibit algal growth, the observed algal growth inhibiting capacity of the test item was maybe caused by the degradation products. However, toxicity of the parent substance cannot be excluded. Therefore, the analytical measurement of test item concentrations was performed at the beginning of the test to check the degradation of the parent substance at the beginning of the exposure. The NOEC/EC-values were reported based on the initial measured concentrations of test item and on the nominal concentration to cover both, the toxicity of the parent and of the degradation products. No other analytical measurements at the end of the test were performed. Due to the high percentage of degradation of the test substance also at test initiation, the effect concentrations selected for the characterisation of the hazard profile of the test substance are based on nominal concentrations, in order to cover the toxicity of the parent substance and the degradation products.

- Test conditions: At the start of the test, the pH measured in the treatments was 8.0. At the end of the test, pH values of 8.1 to 9.2 were measured. The increase of the pH during the test was caused by the uptake of CO2 by the algae due to their rapid growth, despite the test media being stirred during the test. The water temperature during the test was maintained at 22 °C.

- The influence of the test item on the growth of the algae is shown in Table 1 to Table 3 of the field "any other information on results including tables".

- OECD validity criteria:
In the control the biomass increased by a factor of 138 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled.
The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 11%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled.
The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.4%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Any other information on results incl. tables

Table 1      Biomass of Algae

Nominal test item concentration (mg/L)	Initially measured concentration (mg/L)	Rep. no.	Biomass of algae*
			24 hours	48 hours	72 hours
Control	Control	1	9.9	58.7	289.3
		2	9.9	57.1	306.1
		3	9.9	64.5	259.5
		4	9.3	61.8	311.1
		5	9.4	63.7	280.9
		6	9.1	58.3	275.1
		Mean	9.6	60.7	287.0
		SD	0.4	3.1	19.4
0.10	0.033	1	8.7	60.6	288.1
		2	8.9	58.7	300.2
		3	8.5	58.0	295.2
		Mean	8.7	59.1	294.5
		SD	0.2	1.3	6.0
0.32	0.14	1	6.7	50.0	259.0
		2	6.6	50.1	275.4
		3	6.3	46.4	273.4
		Mean	6.5	48.8	269.3
		SD	0.2	2.1	9.0
1.0	0.40	1	2.3	16.8	112.8
		2	2.3	14.7	111.9
		3	3.1	24.3	145.4
		Mean	2.6	18.6	123.3
		SD	0.4	5.0	19.1
3.2	1.4	1	1.2	1.2	1.4
		2	1.2	1.3	1.2
		3	1.5	1.3	1.7
		Mean	1.3	1.3	1.4
		SD	0.2	0.1	0.3
10	5.6	1	1.0	0.7	0.3
		2	1.0	0.8	0.2
		3	0.9	0.6	0.3
		Mean	1.0	0.7	0.2
		SD	0.0	0.1	0.1

 SD:  Standard deviation

*:     The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 10³). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 2.07 x 10³relative fluorescence units.

Table 2      Summary of the biological results (on the basis of initially measured and nominal concentrations of the test item)

	Initially measured		Nominal
Parameter (0-72 h)	Growth rate	Yield	Growth rate	Yield
EC10  (mg/L)	0.36	0.17	0.91	0.39
95% confidence interval	n.d.	0.14 – 0.19	n.d.	0.32 – 0.45
EC20  (mg/L)	0.41	0.22	1.0	0.52
95% confidence interval	n.d.	0.19 – 0.24	n.d.	0.45 – 0.58
EC50  (mg/L)	0.54	0.36	1.3	0.88
95% confidence interval	n.d.	0.34 – 0.38	n.d.	0.83 – 0.94
NOEC (mg/L)	0.14	0.14	0.32	0.32
LOEC (mg/L)	0.40	0.40	1.0	1.0

 n.d.  could not be determined

 

Table 3       Average Growth Rates(µ) and Yield(Y)

Nominal test item concentration	Initially measured concentration	Average growth rate µ (day^-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
(mg/L)	(mg/L)	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	---	1.53	0.0	1.69	0.0	1.64	0.0
0.10	0.033	1.43	6.2	1.67	0.8	1.65	-0.6
0.32	0.14	1.15*	25.0	1.58*	6.4	1.62	1.3
1.0	0.40	0.21*	86.4	1.09*	35.7	1.36*	17.3
3.2	1.4	-0.47*	131.0	-0.24*	114.4	-0.13*	107.8
10	5.6	-0.76*	149.6	-0.57*	133.9	-0.71*	143.4

Nominal test item concentration	Initially measured concentration	Yield Y (x 10^3) and inhibition of Y (Iy)
		0-24 h		0-48 h		0-72 h
(mg/L)	(mg/L)	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	---	7.5	0.0	58.6	0.0	284.9	0.0
0.10	0.033	6.6*	11.6	57.0	2.7	292.4	-2.6
0.32	0.14	4.5*	40.6	46.8*	20.2	267.2	6.2
1.0	0.40	0.5*	93.3	16.5*	71.8	121.3*	57.4
3.2	1.4	-0.8*	110.4	-0.8*	101.4	-0.6*	100.2
10	5.6	-1.1*	114.7	-1.4*	102.4	-1.8*	100.6

 *:          mean value statistically significant lower than in the control (according to Williams t-test, one-sided,a= 0.05)

Note:    Percentageinhibitionvalues in excess of 100% are obtained when the biomass at the end of the interval is lower than at the start of the interval.

 

Table 4       Results Obtained for the Concentrations of the Treatment Samples

Age	Sample ID	Nominal Concentra­tion of Test Item cnom	Measured Concentration  of Test Item x			Sample Preparation Factor F	Determined Average Concentra­tion of Test Item c	Recovery Rate R
			Sample 1	Sample 2	Average
[day]		[mg/L]	[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]
0	A1/2	Control	<LOQana	<LOQana	<LOQana	1.25	<LOQbio	n.a.
	A3/4	0.1	<LOQana	<LOQana	<LOQana	1.25	<LOQbio	n.a.
	A5/6	0.32	0.115	0.114	0.115	1.25	0.143	45
	A7/8	1	0.315	0.321	0.318	1.25	0.398	40
	A9/10	3.2	1.09	1.08	1.09	1.25	1.36	42
	A11/12	10	4.51	4.51	4.51	1.25	5.64	56

LOQ_ana                 =            0.0534 mg test item/L

LOQ_bio                  =            0.0668 mg test item/L

n.a.                        =            not applicable

 The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(OECD validity criteria)

Conclusions:

Based on the results of this study, test item is considered as toxic to the aquatic organisms tested in accordance with the Directive 67/548/EC.

Executive summary:

The influence of the test item HMTBN on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72‑hour static test according to OECD Guideline 201 (2006), the EU Commission Directive 92/69/, C.3 (1992) and the Commission Regulation (EC) No 440/2008, C.3. The study was compliant with the GLP.

 

The nominal concentrations of the test item of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were tested in parallel with a control.

 

The measured concentrations of the test item in the test media of the nominal test concentrations of 0.32 to 10 mg/L were between 40 and 56% of the nominal values at the start of the test. The measured concentration of the test item in the test medium of the nominal test concentration of 0.10 mg/L was below LOQ (Limit of quantification,LOQ_bio: 0.0668 mg/L). During pre-experiments on stability of the test item it has been observed that under the conditions of the algal toxicity test the test item degrades into degradation products. As the degradation products are known to inhibit algal growth, the observed algal growth inhibiting capacity of the test item was maybe caused by the degradation products. However, toxicity of the parent substance cannot be excluded. Considering the kinetic of degradation, analytical measurement of test item concentrations was performed at the beginning of the test only. The effect concentrations are expressed in nominal concentration in order to cover both, the toxicity of the parent and of the degradation products.

 

The results obtained were as follows:

72h-ErC50 = 1.3 mg/L (growth rate)

72h-EbC50 = 0.88 mg/L (biomass)

72h-NOErC = 0.32 mg/L (growth rate and biomass)

Based on these results, HMTBN should be classified as toxic to the aquatic organisms tested in accordance with the Directive 67/548/EC.

 

The three validity criteria of the OECD guideline 201 were fulfilled. But due to the uncertainties on the concentrations of the parent and degradation products tested, this study is considered as reliable with restrictions.