2-methylglutaronitrile

Phototransformation in water

Administrative data

Endpoint:

phototransformation in water

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study does not follow a particular guideline and is not GLP compliant. However, it is well described both in terms of experimental conditions and results, and allows to scientifically conclude on the results obtained during the algal study.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 31 MARCH TO 23 AUGUST 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study follows an internationaly recognized guideline (OECD 201) and is GLP compliant.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 32, 100, 316, 1000 mg/L
- Sampling method: No data.
- Sample storage conditions before analysis: all samples were stored in a freezer (=< -10°C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test item concentration of 1000 mg test item/L was prepared by dissolving 500 mg test item into 500 mL test water by intense stirring for 15 minutes. The pH of this solution was adjusted with 0.1 M NaOH to pH 8.1. Adequate volumes of this solution were diluted with test water to prepare the test media of the other desired test concentations. The test media were prepared just before introduction of the algae.
- Controls: Test water without addition of test item.
- Blanks: One replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. the additional replicates were incubated under the same conditions as the others.
No further data.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologishces Institut der Universität Göttingen, 37073 Göttingen, Germany
- Age of inoculum (at test initiation): no data
- Method of cultivation: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: The cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L = 24 mg/L as CaCO3

Test temperature:

22-23°C

pH:

from 8.0-8.1 at the beginning of the test, to 8.4-9.6 at the end of the test.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

- nominal conc.: 0, 10, 32, 100, 316, and 1000 mg/L
- measured concentrations at 0h: 0, 9.17, 31.64, 104.90, 296.15, 1064.00 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: erlenmeyer flasks of 50 mL volume
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: erlenmeyer flasks of 50 mL volume filled with 50 mL of test medium
- Aeration: no data
- Initial cells density: 5000 cells/mL
- Control end cells density: 793970 cells/mL after 72 hours in control
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: analytical grade salts were added in deionised water.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity < 5µScm-1
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured in all test item concentrations and the control at the start and the end of the test. The appearance of the test item in test water was determined daily in all test item concentrations.

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: with 0.1 M NaOH in the stock solution
- Photoperiod: continuous (24:0)
- Light intensity and quality: 5892 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell densities in the samples were determined by spectrophotometrical measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure. The algal cell densities were calculated by substracting the absoprtion of the blanks, from each of the measured absorption of the test media. The cells densities in a number of samples from one control replicate were counted by microscope after 72 hours. Out of this control five defined dilutions were prepared and measured spectrophotometrically. Based on the counted cell densities and the absorption of the control sample and the dilutions a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrically during the test. The inhibition of algal growth was detrmined from: (i) the average specific growth rate, (ii) the yield = the mean cell density at the end of the test minus the mean cell density at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3.2
- Range finding study
- Test concentrations of the range finding study: limit test at 1000 mg/L
- Results used to determine the conditions for the definitive study: no growth inhibition during the preliminary limit test at 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 080 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 926-1391

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 228.67 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 387 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 178-522

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 440.27 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 100 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 113.77 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 316 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 359.5 mg/L

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 463 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 400-543

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 526.73 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 199 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: 141-245

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 226.39 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 11.38 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 36.41 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Details on results:

Since 90% and 0% of nominal concentration of MGN were measured at 0 and 72h respectively, an additional test monitoring the test substance for 72 hours under light and obscurity conditions, with or without algae, was conducted (see endpoint sumary record in 5.1). The results confirmed that results of the current study could be based on nominal concentrations (please refer to IUCLID section 5.1.3).

Results with reference substance (positive control):

- 72h-EC50= 2.305 mg/L (last test performed in 2010)
No further data.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC10 values and where possible their 95%-confidence limits were calculated by Probit analysis.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by the Welch t-test (growth rate) and the William t-test (yield), respectively.
Based on the results of this statistical evaluation, the 72-hour NOEC and LOEC for growth rate were not determinable. Therefore, both values were directly determined by visual inspection of the raw data.
Statistics were performed using ToxRat Professional Version 2.10, ToxRat (R) Solutions GmbH.

Table: Results for the determination of the test item in the test samples.

Sample description			test item		calculated
	age		found		test item	nominal	% of
[mg/L]	day	[h]	[mg/L]	D.F.	[mg/L]	[mg/L]	nominal
control	0	0	<LOD	1	n.a.	0.000	n.a.
control	3	72	<LOD	1	n.a.	0.000	n.a.
10	0	0	9.143	1	9.143	10.000	91
10	0	0	9.198	1	9.198	10.000	92
10	3	72	<LOD	1	n.a.	10.000	n.a.
10	3	72	<LOD	1	n.a.	10.000	n.a.
32	0	0	31.593	1	31.593	32.000	99
32	0	0	31.68	1	31.680	32.000	99
32	3	72	<LOD	1	n.a.	32.000	n.a.
32	3	72	<LOD	1	n.a.	32.000	n.a.
100	0	0	10.639	10	106.390	100.000	106
100	0	0	10.342	10	103.420	100.000	103
100	3	72	<LOD	10	n.a.	100.000	n.a.
100	3	72	<LOD	10	n.a.	100.000	n.a.
320	0	0	30.467	10	304.670	320.000	96
320	0	0	28.763	10	287.630	320.000	91
320	3	72	<LOD	10	n.a.	320.000	n.a.
320	3	72	<LOD	10	n.a.	320.000	n.a.
1000	0	0	12.102	100	1210.200	1000.000	121
1000	0	0	9.178	100	917.800	1000.000	92
1000	3	72	<LOD	100	n.a.	1000.000	n.a.
1000	3	72	<LOD	100	n.a.	1000.000	n.a.

Validity criteria fulfilled:

yes

Conclusions:

Based on nominal concentrations, the toxicity of the test substance on algae growth rate was calculated as ErC50(72h) = 1080 mg/L, NOErC(72h) = 100 mg/L and the associated LOErC(72h) = 316 mg/L.

Executive summary:

Toxicity of the substance to Pseudokirchneriella subcapitata has been tested in an algal growth inhibition test, following OECD guideline 201 and EC N°761/2009 C.3 under GLP conditions.

A stock solution of 1000 mg/L was prepared by direct addition into test water. Five different test concentrations (10, 32, 100, 316, and 1000 mg/L) were prepared in triplicates by serial dilution into OECD test water. Six replicates of control solution were prepared only with test water. At the beginning of the test 5 000 cell/mL were inoculated in each test vessels and light intensity was set at an average of 5892 lux. Chemical monitoring were performed through a dedicated UV-HPLC method at 0H and 72h in each test groups.

After 72 hours of exposure, no test substance was measured in the test groups. Thus an additional study was conducted in order to monitor the test substance, through UV-HPLC and TOC analyses, at 0h, 24h, 48h, and 72h under light/no light and algae/no algae conditions. This additional study showed TOC remained constant throughout the experimental phase. However, as measured by UV-HPLC, the test substance decreased showing evidence for a transformation during the test. As results were the same under both light conditions and only occurred when algae were present, external metabolization by algae is assumed without photodegradation.

This observation figured out to base algae results on nominal concentrations (see corresponding endpoint summary record).

Based on nominal concentrations, the toxicity of the test substance on algae growth rate was calculated as ErC50(72h) = 1080 mg/L, NOErC(72h) = 100 mg/L and the associated LOErC(72h) = 316 mg/L.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Study type:

other: photodegradation during an OECD 201 algal test

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Test design:
- 2 concentrations were tested: 100 and 500 mg/L
- 1 replicate with algae in the light (per conc.)
- 1 replicate with algae in the dark (per conc.)
- 1 replicate without algae in the light (per conc.)
- 1 replicate without algae in the dark (per conc.)
- Each replicate was divided in 5 test beakers with 50 mL each
- Light exclusion was achieved by completely coverage of the test beakers in the dark. They were completely wrapped in aluminium foil.
- All beakers were incubated at exactly the same conditions used in a normal Algae test
- Sampling after 0, 24, 48, and 72h
- Per sampling one beaker of each replicate was sacrificed (except at 0h where only one beaker representative for all replicates was sacrificed)
- Standby analysis of the test item after 0, 24, 48, 72 h with HPLC
- calibration with test item
- fortified samples with test item
- Standby analysis of total carbon content after 0, 24, 48, 72 h with TOC
- calibration with test item
- fortified samples with test item

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methylglutaronitrile

EC Number:

224-923-5

EC Name:

2-methylglutaronitrile

Cas Number:

4553-62-2

Molecular formula:

C6H8N2

IUPAC Name:

2-methylpentanedinitrile

Study design

Radiolabelling:

no

Analytical method:

high-performance liquid chromatography

other: Total Organic Carbon (TOC) analysis

Details on sampling:

- Sampling intervals: Both 2-methylglutaronitrile (MGN) and TOC were analysed after 0, 24, 48 and 72 hours.
- Sampling method: Each replicate of each treatment was divided in 5 test beakers intended to perform the analyses at the different times. Per sampling time, one beaker of each replicate was sacrificed (except at 0h where only one beaker reprsentative for all replicates was sacrificed).
No further data.

Buffers:

No data

Light source:

other: As for the algae study (OECD 201)

Details on light source:

Neon ligth tubes as for a typical algae study (OECD 201). For futher details on light conditions, please refer to IUCLID section "6.1.5 Toxicity to aquatic algae and cyanobacteria" and the ESR relative to the study of Hoffmann & Deierling (2010).

In the case of treatment performed in the dark, light exclusion was achieved by completely coverage of the test beakers in the dark. They were completely wrapped in aluminium foil.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test apparatus/vessels: 50 mL test beakers
No further data.

TEST MEDIUM
The same medium as in the algal test was used. For futher details, please refer to IUCLID section "6.1.5 Toxicity to aquatic algae and cyanobacteria" and the ESR relative to the study of Hoffmann & Deierling (2010).

REPLICATION
- No. of replicates: 8 different treatments were applied:
* 100 mg/L MGN - With Algae - In the light: 1 replicate
* 100 mg/L MGN - With Algae - In the dark: 1 replicate
* 100 mg/L MGN - Without Algae - In the light: 1 replicate
* 100 mg/L MGN - Without Algae - In the dark: 1 replicate
* 500 mg/L MGN - With Algae - In the light: 1 replicate
* 500 mg/L MGN - With Algae - In the dark: 1 replicate
* 500 mg/L MGN - Without Algae - In the light: 1 replicate
* 500 mg/L MGN - Without Algae - In the dark: 1 replicate

Duration of test at given test condition

Duration of test at given test condition 1

Duration:

72 h

Initial conc. measured:

100 mg/L

Duration of test at given test condition 2

Duration:

72 h

Initial conc. measured:

500 mg/L

Reference substance:

no

Dark controls:

yes

Computational methods:

No data

Results and discussion

Transformation products:

not specified

Details on results:

TOC analysis:
Regardless the treatment applied (100 or 500 mg/L, light or darkness, algae or not), the TOC remained constant over time.

HPLC analysis of MGN:
Regardless the initial concentration and the light conditions, a decrease in the MGN concentration over time was observed when the test occurred in the presence of algae. The rate of disappearance reached 100% at the end of the 72-hour exposure (except for the treatment with an initial concentration of 500 mg/L and no light). In the absence of algae, such decrease was not observed and the MGN concentrations remained stable over time.

Any other information on results incl. tables

Table: Results for the determination of the test item in the test samples (HPLC)

Sample description					test item		calculated
	age				found		test item	nominal	% of
[mg/L]	day	[h]	light	algae	[mg/L]	D.F.	[mg/L]	[mg/L]	nominal
100	0	0			98.308	1	98.308	100.000	98
100	1	24	X	X	90.789	1	90.789	100.000	91
100	1	24		X	100.663	1	100.663	100.000	101
100	1	24	X		94.206	1	94.206	100.000	94
100	1	24			100.874	1	100.874	100.000	101
100	2	48	X	X	-1.851	1	-1.851	100.000	-2
100	2	48		X	-1.831	1	-1.831	100.000	-2
100	2	48	X		101.379	1	101.379	100.000	101
100	2	48			101.477	1	101.477	100.000	101
100	3	72	X	X	0.137	1	0.137	100.000	0
100	3	72		X	0.233	1	0.233	100.000	0
100	3	72	X		99.24	1	99.240	100.000	99
100	3	72			100.422	1	100.422	100.000	100
500	0	0			100.517	5	502.585	500.000	101
500	1	24	X	X	101.716	5	508.580	500.000	102
500	1	24		X	101.844	5	509.220	500.000	102
500	1	24	X		102.765	5	513.825	500.000	103
500	1	24			102.495	5	512.475	500.000	102
500	2	48	X	X	83.827	5	419.135	500.000	84
500	2	48		X	99.049	5	495.245	500.000	99
500	2	48	X		103.703	5	518.515	500.000	104
500	2	48			103.34	5	516.700	500.000	103
500	3	72	X	X	0	5	0.000	500.000	0
500	3	72		X	90.248	5	451.240	500.000	90
500	3	72	X		101.399	5	506.995	500.000	101
500	3	72			101.735	5	508.675	500.000	102

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Conclusions:

The substance appears to be metabolized by algae and no photo-transformation seems to occur. This is the reason why the TOC remains constant.

Executive summary:

Due to the disappearance of the substance in a previous algae study (Hoffmann & Deierling, 2010), a specific analytical experiment was conducted in order to understand the fate of the test substance in the study and conclude scientifically.

Two different concentrations of the substance were tested (100 and 500 mg/L), with or without algae, in the light or in the dark (1 replicate for each concentration, for each experimental condition); every other experimental conditions remaining the same as for the algae test (Hoffmann & Deierling, 2010). The test substance was monitored by UV-HPLC as detailed in Hoffmann & Deierling (2010) along with total organic carbon (TOC) after 0, 24, 48, and 72 h. Total organic carbon remained constant throughout the test; showing the test substance was still in the test water. However, as measured by UV-HPLC, the test substance decreased showing evidence for a transformation during the test. As results were the same under both light conditions and only occurred when algae were present, external metabolization by algae is assumed without photodegradation.

The results suggest therefore that algae metabolized the test substance whatever the light conditions and that the test item remained in the medium. Thus algae results could be based on nominal concentrations.