Oleonitrile

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:

2010-01-29 to 2010-05-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Oleonitrile

EC Number:

204-016-0

EC Name:

Oleonitrile

Cas Number:

112-91-4

Molecular formula:

C18H33N

IUPAC Name:

(9Z)-octadec-9-enenitrile

Details on test material:

- Name of test material (as cited in study report): Oleylnitrile (CAS No. 112-91-4)
- Physical state: Liquid, light yellow
- Composition of test material, percentage of components: 101.1 % (w/w), active content nitriles
- Purity test date: 2009-09-21
- Lot/batch No.: S001149
- Expiration date of the lot/batch: 2018-01-30
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of loadings all loading levels and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates of the second lowest and the second highest test loading (0.01 and 3.20 mg/L) was prepared without algae for analysis after 72 h to demonstrate stability of the test item. Analysis after 72 h of these loadings were carried out. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a loading level close to the EL50-value (the second highest test loading of 3.20 mg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at 6 ± 2°C until start of analysis, if necessary. The prepared samples were stored on the autosampler until measuring.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (10.0 mg/L was weighed out) was prepared with test medium and shaken with 20 rpm for 24 hours (WSF) at room temperature. After a separation phase of 24 hours the WSF was taken from the homogeneous liquid phase.
- Eluate: Natural water
- Differential loading: The water soluble fraction (WSF) was tested and further seven dilution levels out of the WSF with a dilution factor √10: 0.00320 - 0.0100 - 0.0320 - 0.100 - 0.320 - 1.00 - 3.20 and 10.0 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDAK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 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): Not the same, standard growth medium
- 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:

After 72 h of exposure aliquots of the test medium (i.e. samples of exposed algal cells) from test loadings 0.032 to 10.0 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 3 days under test conditions. The test item effect was observed to be reversible at these loading. Therefore, there is potential for recovery following exposure up to 10.0 mg/L (highest test loading).
For details please refer to the table under information on materials and methods

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Nominal test item loading Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
10.0 4.79 7.71 7.44
3.20 1.74 7.73 7.47
1.00 0.538 7.87 7.48
0.320 0.123 7.92 7.48
0.100 0.0407 7.93 7.53
0.0320 0.0129 7.96 7.58
0.0100 0.00561 7.96 7.86
0.00320 0.00175 7.96 7.91
Control 7.96 8.00

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with cotton wool plugs
- 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: 10058 cells/mL
- Control end cells density: Mean 1164504 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: Water of the river Leine was used. This river is located near
D-31171 Nordstemmen, Marienbergstraße, Germany (52° 10’ 14.98‘’, 9° 46’ 7.64’’). Water parameters are given in Table 1. The water was filtered with a membrane filter (0.45 µm, RC, MACHERY-NAGEL). Additionally 50 % of the components concentrations of the dilution water (total application volume 6.5 mL/L) acc. to the guideline were added to enable a sufficient growth of algae

Characterisation Data of Natural River Water
River Leine
Location D-31171 Nordstemmen, Marienbergstrasse
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. - 1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
o-Phosphate-P [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154


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)

Nominal Loading of the Water Accommodated Fraction (WAF)
[mg/L] Specific Growth Rate Inhibition[%] Yield Inhibition
[%]
10.0 100 100
1.00 56 96
0.10 7 35
0.01 2 13
0.001 3 15

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Effect conc.:

8.57

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (6.75 - 10.7)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.107

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.0817-0.148)

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: 0.74 (CI 0.71 - 0.78) mg/L
Yield Inhibition: 0.36 (CI < 0.35 - 0.36) mg/L

Reported statistics and error estimates:

EL10-, EL20- and EL50-values of the growth rate and yield
analyses inhibition after 72 h were calculated by sigmoidal dose-response regression and polynomial (fourth order) regression, respectively. Calculation of the confidence intervals of EL10-, EL20- and EL50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Any other information on results incl. tables

EL₁₀-_, EL₂₀- and EL₅₀- values and 95 % Confidence Intervals of
Oleylnitrile(CAS No. 112-91-4) (0-72 h)

                                based on initial measured test item concentrations [mg/L]

                               

	Rate-related inhibition
ErL10	0.00857 (0.00675 – 0.0107)
ErL20	0.0152 (0.0121 – 0.0195)
ErL50	0.107 (0.0817 – 0.148)
	Inhibition of yield
ErL10	0.00561
ErL20	0.00625 (0.00597 – 0.00652)
ErL50	0.0114 (0.0107 – 0.0121)

Cell Densities

	Nominal test item loading		Initial measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]				No.		0 h		24 h	48 h	72 h
10.0		4.79		1		10058		16483	16663	25082
				2		10058		16890	17613	27313
				3		10058		16952	17979	28245
				Mean		10058		16775	17418	26880
3.20		1.74		1		10058		17743	22684	63294
				2		10058		17822	22236	49674
				3		10058		18227	21427	43248
				Mean		10058		17931	22116	52072
1.00		0.538		1		10058		22640	27077	62211
				2		10058		20386	23590	49494
				3		10058		18415	19651	41493
				Mean		10058		20480	23439	51066
0.320		0.123		1		10058		25932	35071	88068
				2		10058		20864	37233	87327
				3		10058		20967	32171	76333
				Mean		10058		22588	34825	83909
0.100		0.0407		1		10058		27277	67226	219260
				2		10058		26746	67651	242070
				3		10058		30351	85088	245483
				Mean		10058		28125	73322	235604
0.0320		0.0129		1		10058		34378	117981	447534
				2		10058		35425	126459	546427
				3		10058		34708	136648	522969
				Mean		10058		34837	127029	505643
0.0100		0.00561		1		10058		39590	136970	1019350
				2		10058		39706	161128	999134
				3		10058		41449	163954	1039404
				Mean		10058		40248	154017	1019296
0.00320		0.00175		1		10058		38045	134076	983098
				2		10058		38446	125789	1047464
				3		10058		37785	142171	1000680
				Mean		10058		38092	134012	1010414
Control				1		10058		40148	165868	1187598
				2		10058		43080	163393	1207563
				3		10058		38691	165724	1089746
				4		10058		38543	142805	1181453
				5		10058		41478	150142	1076580
				6		10058		42835	189896	1244084
				Mean		10058		40796	162971	1164504

Evaluation after 72 h

             

Nominal test item loading	Initial measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]		No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	4.79	1		0.305	80.8		15024	98.7
		2		0.333	79.0		17255	98.5
		3		0.344	78.3		18187	98.4
		Mean		0.327	79.3		16822	98.5
3.20	1.74	1		0.613	61.3		53236	95.4
		2		0.532	66.4		39616	96.6
		3		0.486	69.3		33190	97.1
		Mean		0.544	65.7		42014	96.4
1.00	0.538	1		0.607	61.6		52153	95.5
		2		0.531	66.5		39436	96.6
		3		0.472	70.2		31435	97.3
		Mean		0.537	66.1		41008	96.5
0.320	0.123	1		0.723	54.3		78010	93.2
		2		0.720	54.5		77269	93.3
		3		0.676	57.3		66275	94.3
		Mean		0.706	55.4		73851	93.6
0.100	0.0407	1		1.03	35.1		209202	81.9
		2		1.06	33.0		232012	79.9
		3		1.07	32.7		235425	79.6
		Mean		1.05	33.6		225546	80.5
0.0320	0.0129	1		1.27	20.1		437476	62.1
		2		1.33	15.9		536369	53.5
		3		1.32	16.8		512911	55.6
		Mean		1.31	17.6		495585	57.1
0.0100	0.00561	1		1.54	2.77		1009292	12.6
		2		1.53	3.19		989076	14.3
		3		1.55	2.36		1029346	10.8
		Mean		1.54	2.77		1009238	12.6
0.00320	0.00175	1		1.53	3.54		973040	15.7
		2		1.55	2.20		1037406	10.1
		3		1.53	3.16		990622	14.2
		Mean		1.54	2.97		1000356	13.4
Control		1		1.59			1177540
		2		1.60			1197505
		3		1.56			1079688
		4		1.59			1171395
		5		1.56			1066522
		6		1.61			1234026
		Mean		1.58			1154446

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Oleylnitrile (CAS No. 112-91-4) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (initial measured test item concentrations): The EL10-values with 95 % confidence intervals for inhibition of specific growth rate (ErL10) and yield (EyL10) after 72 h were 0.00857 (0.00675 – 0.0107) and < 0.00561 mg/L, respectively. The EL50-values with 95 % confidence intervals for inhibition of specific growth rate (ErL50) and yield (EyL50) after 72 h were 0.107 (0.0817 – 0.148) and 0.0114 (0.0107 – 0.0121) mg/L, respectively.

The concentrations of Oleylnitrile (CAS No. 112-91-4) were analysed at all concentration levels at the start and the end (from two concentrations) of the study via GC-MS analysis. The measured concentrations at test start were in the range of < LOQM – 56 % of the nominal values. At the end of the test Oleylnitrile (CAS No. 112-91-4) was analysed at the concentration levels 0.01 and 3.20 mg/L (prepared without algae) and gave recoveries of < LOQM- 8 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease is therefore attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. Measurements from the glassware (adsorption sample) after a process of concentrating
(s. Preparation of Adsorption sample) were determined below LOQS. So it can be concluded the sorbed amount of test item was < 0.625 % of the applied amount. Therefore all effect values are given based on nominal concentrations of the test item.

The test item effect was observed to be reversible at 0.0320 to 10.0 mg/L (corresponding to initial measured test item concentrations of 0.0129 to 4.79 mg/L). Therefore, there is potential for recovery following exposure up to 4.79 mg/L (highest test loading). All effect levels are given based on initial measured concentrations of Oleylnitrile (CAS No. 112-91-4).

Executive summary:

The toxicity of Oleylnitrile (CAS No. 112-91-4) (Batch no.: S001149) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from January 29 to May 26, 2010 with the definitive exposure phase from May 18 to 21, 2010. The aim of the study was the determination of EL₁₀ - , EL₂₀- and EL₅₀ - values of growth rate and yield over a period of 72 h.

Oleylnitrile (CAS no. 112-91-4) is insoluble in water and also has a strong tendency to adsorb to suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many poorly soluble substances in general but long chain nitriles in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for substances like the nitriles as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with these substances.

The aquatic ecotoxicity tests with nitriles were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulkis compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

         

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

         

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment.

An individual test design was applied. Natural river water was used as dilution medium

The study was conducted under static conditions with an initial cell density of approximately 1 x 10⁴ cells/mL. One water soluble fraction (WSF) and seven further dilution levels, with nominal loadings of 0.00320 - 0.0100 - 0.0320 - 0.100 - 0.320 - 1.00 - 3.20 and 10.0 mg/L were tested in a geometrical series with a dilution factor of √10. Three replicates were tested for the test item loadings and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Oleylnitrile were analysed at all loading levels (prepared without algae) and the control at test start via GC-MS analysis. The measured concentrations at test start were in the range of LOQ – 56 % of the nominal values. At the end of the test Oleylnitrile was analysed at loading levels 0.0100 and 3.20 mg/L (prepared without algae) and gave recoveries of LOQ and 8 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, also the river water was frozen before use to minimize the microbial activity. The decrease is therefore attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. No test item could be recovered from the glassware ( LOQ and 0.625 % of the applied amount) which means that the test organisms were fully exposed to the initial observed test concentrations. Therefore all effect values are given based on initial measured concentrations of the test item. For details of the analytical results please refer to part 9.

Oleylnitrile (CAS No. 112-91-4) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (initial measured test item concentrations): The EL₁₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rL₁₀) and yield (E_yL₁₀) after 72 h were 0.00857 (0.00675 – 0.0107) and 0.00561 mg/L, respectively. The EL₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rL₅₀) and yield (E_yL₅₀) after 72 h were 0.107 (0.0817 – 0.148) and 0.0114 (0.0107 – 0.0121) mg/L, respectively.

After 72 h algae were transferred from the control and the nominal loadings of 0.0320 to  10.0 mg/L (corresponding to initial measured test item concentrations of 0.0129 to 4.79 mg/L) to fresh untreated medium and allowed to grow for further 3 d under test conditions. The test item effect was observed to be reversible at these loadings. Therefore, there is potential for recovery following exposure up to the initial measured test item concentration of 4.79 mg/L (highest test loading).