Fatty acids C18 unsat, reaction products with tetraethylenepentamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline studies, no chemical analysis, not conducted to GLP, no certificates of analysis additional DOC added to test media. Reliable as supporting evidence only.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Tests were conducted in media with 2mg/L DOC added as Humic acid.

Principles of method if other than guideline:

150 mg/L of NaHCO3 was used to aid pH stabilization and 2mg/L DOC as Humic acid was added to the further standard OECD algae media.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No Data

Analytical monitoring:

no

Details on sampling:

No chemical analysis was conducted

Vehicle:

no

Details on test solutions:

A stock solution of approximately 10 - 15 mg/L of each of the test substances was prepared by adding an accurately measured amount of 0.01-
0.015 g of each test material, weighed out on an analytical balance, to 1000 mL of OECD medium containing 2 mg/L DOC as humic acid. These
solutions were agitated thoroughly until a homogeneous solution was obtained. Extra care was also taken to ensure that the humic acid added was also fully dissolved. Stirring time was up to one hour. The pH of the stock solution was checked and found to be between 8.2 and 8.5 in all cases.
The stocks were therefore not adjusted for pH.

The test solutions were prepared by addition of adequate amounts of stock solution (taking into account the slight variation from the different
stocks) to the test vessels, then dilution water (containing humic acid) was added up to the required final volume. The following final nominal test
concentrations for all test substances were prepared: 0.02, 0.06, 0.18, 0.54, and 1.62 mg/L. Controls containing only test medium and test medium + humic acid were included in the test. All test concentrations were agitated for one hour in the orbital shaker prior to inoculation to allow
reaction with the humic acid. Due to poor solubility of some the test substances the following additional test concentrations were tested for
Asco 60, Wetfix AP17, Imidazoline 9711X and Emulsamine 740: 0.03, 0.097, 0.312, 1.0 and 3.2 mg/L. The preparation method was not otherwise
altered for these repeated test concentrations.

Test organisms (species):

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

Details on test organisms:

The tests were carried out with the freshwater unicellular algae P. subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and
Protozoa, Dunstaffnage Marine Laboratory, Oban, Argyll, United Kingdom. After purchasing this strain was cultured and maintained. Cultures on
sloped agar tubes were stored at 4°C until required.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

2 mg/L DOC as Humic acid added to test media

Post exposure observation period:

Control replicates observed for bacterial contamination at the end of the study. No contamination of the algae cultures was observed.

Hardness:

Not measured. OECD test media used with 150 mg/L NaHCO3 and 2 mg/L additional DOC as humic acid.

Test temperature:

The temperature varied 23 ± 2 °C during the tests.

pH:

The pH measurements in Table 12 – 21 show a maximum increase of 1.9 pH units.

Dissolved oxygen:

Not measured

Salinity:

Not measured.

Nominal and measured concentrations:

0.02, 0.06, 0.18, 0.54, and 1.62 mg/L or 0.03, 0.097, 0.312, 1.0 and 3.2 mg/L. (Nominal concentrations)

Details on test conditions:

All light intensity measurements were > 96 µmol •m-2•s-1 and < 120 µmol• m-2•s-1

Reference substance (positive control):

yes

Remarks:

Conducted in laboratory as part of GLP maintainance. Culture performance acceptable

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

1: CAS no: 1226892-43-8

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.88 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

2: CAS no 1224966-15-7

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

3: CAS no 1224966-13-5

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

4: CAS no 1226892-45-0

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

5: CAS no 1226892-50-7

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

6: CAS no 68991-84-4

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

7: CAS no 93572-25-9

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

8: CAS no 84082-48-4

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

9: CAS no 68910-93-0

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

10: CAS no 91001-82-0

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

1: CAS no 1226892-43-8

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

2: CAS no: 1224966-15-7

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated*

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

3: CAS no 1224966-13-5

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

4: CAS no 1226892-45-0

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated*

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

5: CAS no 1226892-50-7

Basis for effect:

growth rate

Remarks on result:

other: linear Interpolation

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

6: CAS no 1226892-46-1

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

7: CAS no 1226892-49-4

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.41

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

8: CAS no 84082-48-4

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated*

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.55 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

9: CAS no 68910-93-0

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated*

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

10: CAS no 91001-82-0

Basis for effect:

growth rate

Remarks on result:

other: Extrapolated*

Details on results:

All tests were conducted with 2.0 mg/L DOC as Humic acid added to the test media to allow a more environmentally realistic endpoint to be generated as definitive tests were to be conducted according to the bulk approach in natural water. Extrapolated values should be interpreted with care.

Results with reference substance (positive control):

The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when the cultures are cultured from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, which means that the EC50 values are between 0.25 and 2.0 mg/L.

Reported statistics and error estimates:

The mean values of the absorbance’s for each test substance concentration were used to calculate the growth and specific growth rate. Where
possible, the EC10, and 50 values were computed from the best fitted line (least-squares method) through the points given by the probit of the
percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50, whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC)
was determined by comparison of the growth at each concentration and the control using threshold values from the Dunnetts or William’s tests . The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no
significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller’s theorem if possible . All computations
were performed using the TOXCALC version 5.0 program. All results in this report are based on growth rate.

Study conducted as a series of range finding tests for subsequent definitive tests. OECD guidelines were followed and methodology can be considered reliable. No chemical analysis and No GLP accreditation was given and test media was adapted slightly. Study was conducted in a GLP laboratory however.

Validity criteria fulfilled:

yes

Conclusions:

Results may be considered reliable as supporting evidence only.

Executive summary:

Study performed according to the most recent OECD 201 guideline. Study is performed without GLP administration but following exactly the same procedures. Tests were performed without chemical analysis mainly to compare the algae toxicity for different products focussing on obtaining ErC10 values also in relation to the limited solubility of the test substances. Most tests batches used were in a later stage also used for GLP studies. Some of the ErC50 values were derived using extrapolation. These may not be realistic in relation to the limited solubility of these test substances. Extrapolated values should therefore be interpreted with care.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Remarks:

This study is a Key study for Risk assessment.

Adequacy of study:

key study

Study period:

2010-03-01 to 2010-03-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses. Test performed forthe purpose of performing risk assessment according to the Bulk approach is considered less suitable for C&L.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: From the series of concentrations the second lowest and the second highest test concentration (0.032 and 3.20 mg/L) and the control were analytically verified at the beginning of the test.
- Sampling method: Separate replicates for the test item analysis after 0 and 72 h were prepared without algae. Analysis after 72 h of these concentrations were carried out. Sorption to the walls of the glass container was checked out of one test replicate (prepared with algae) of a concentration level close to the EC50-value (the second highest test concentration of 3.20 mg/L was analysed).
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A sotck solution of 10 mg/L was freshly prepared with test medium. Dispersion treatment was 5 min ultrasound at room temperature and equilibration phase for 1 h before application.
- Eluate: Natural river water
- Differential loading: 0.010 - 0.032 - 0.100 - 0.320 - 1.00 - 3.2 - 10 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

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): 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): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h of exposure, 0.2 – 1 mL aliquots of the test medium (i.e. samples of exposed algal cells) from the test concentrations 1.00 – 3.20 mg/L and control were transferred to 10 mL untreated test medium. Algae were then allowed to grow for further 4 – 12 days under test conditions. The test item effect was observed to be reversible at 1.00 mg/L and not reversible at 3.2 mg/L. Therefore, there is potential for recovery following exposure up to 1.00 mg/L (third highest test concentration).
For details please refer to "Any other information on material and methods incl. tables".

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
10.0 8.11 7.96
3.20 7.90 7.98
1.00 7.90 8.00
0.320 7.91 8.78
0.100 7.90 8.93
0.032 7.92 9.03
0.010 7.92 9.15
Control 7.90 9.16

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other 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: 9787 cells/mL
- Control end cells density: Mean 3164375 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. 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
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
10.0 100 100
1.00 31 80
0.10 6 28
0.01 1 4
0.001 - 1 - 7

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.395 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.356 – 0.452)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.638 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (0.589 - 0.683)

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: 0.82 (CI 0.78 - 0.85) mg/L
Yield Inhibition: 0.37 (CI 0.36 - 0.40) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

EC₁₀-_, EC₂₀-and EC₅₀- values and 95 % Confidence Intervals of
Tall oil reactionproducts with tetraethylenepentamine (CAS No. 68555-22-6)             (0-72 h)

                                based on nominal test item concentrations [mg/L]

                               

	Rate-related inhibition
ErC10	0.395 (0.356 – 0.452)
ErC20	0.478 (0.433 – 0.526)
ErC50	0.638 (0.589 – 0.683)
	Inhibition of yield
EyC10	≤ 0.010
EyC20	0.291 (0.195 – 0.621)
EyC50	0.398 (0.330 – 0.683)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
10.0		1		9787		-130	-699	0
		2		9787		11	39	0
		3		9787		4171	1562	0
		Mean		9787		1351	301	0
3.20		1		9787		688	453	0
		2		9787		1966	495	1900
		3		9787		2041	918	1900
		Mean		9787		1565	622	1267
1.00		1		9787		20508	16353	14036
		2		9787		26134	21222	20531
		3		9787		22092	18990	17504
		Mean		9787		22911	18855	17357
0.320		1		9787		61816	355416	2284620
		2		9787		61407	467910	2411623
		3		9787		67235	480093	2374254
		Mean		9787		63486	434473	2356832
0.100		1		9787		60242	505891	2435246
		2		9787		70201	620717	3018370
		3		9787		69717	578548	2795956
		Mean		9787		66720	568385	2749857
0.032		1		9787		67376	519789	2671858
		2		9787		64016	457674	2696147
		3		9787		75874	618221	2926908
		Mean		9787		69089	531895	2764971
0.010		1		9787		69533	607096	2925841
		2		9787		48755	430493	2590938
		3		9787		70304	550005	2926414
		Mean		9787		62864	529198	2814398
Control		1		9787		72762	615297	3016180
		2		9787		75084	668873	3141035
		3		9787		78214	606555	3203968
		4		9787		73058	632673	3217283
		5		9787		80127	636128	3128392
		6		9787		80973	652423	3279389
		Mean		9787		76703	635325	3164375

Evaluation after 72 h

             

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1		0.000	100		-9787	100
	2		0.000	100		-9787	100
	3		0.000	100		-9787	100
	Mean		0.000	100		-9787	100
3.20	1		0.000	100		-9787	100
	2		-0.546	100		-7887	100
	3		-0.546	100		-7887	100
	Mean		-0.364	100		-8520	100
1.00	1		0.120	93.8		4249	99.9
	2		0.247	87.2		10744	99.7
	3		0.194	89.9		7717	99.8
	Mean		0.187	90.3		7570	99.8
0.320	1		1.82	5.63		2274833	27.9
	2		1.84	4.70		2401836	23.9
	3		1.83	4.97		2364467	25.1
	Mean		1.83	5.10		2347045	25.6
0.100	1		1.84	4.53		2425459	23.1
	2		1.91	0.810		3008583	4.63
	3		1.89	2.14		2786169	11.7
	Mean		1.88	2.49		2740070	13.1
0.032	1		1.87	2.92		2662071	15.6
	2		1.87	2.77		2686360	14.8
	3		1.90	1.34		2917121	7.53
	Mean		1.88	2.34		2755184	12.7
0.010	1		1.90	1.35		2916054	7.56
	2		1.86	3.45		2581151	18.2
	3		1.90	1.35		2916627	7.54
	Mean		1.89	2.05		2804611	11.1
Control	1		1.91			3006393
	2		1.92			3131248
	3		1.93			3194181
	4		1.93			3207496
	5		1.92			3118605
	6		1.94			3269602
	Mean		1.93			3154588

Validity criteria fulfilled:

yes

Conclusions:

The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to ascertain a realistic exposure of the test organisms and to quantify the extent of this exposure the results are considered to be reliable. Finally, the test result is considered adequate for the evaluation of the environmental risks in the EU applying the bulk approach considering the realistic worst case (low) concentration of suspended matter (16.2 mg/L) and DOC (3.9 mg C/L) in the river water used.

Executive summary:

The toxicity of Tall oil reaction products with tetraethylenepentamine (CAS No. 68555-22-6) (Batch no.: S000925)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 March 01 to 16, 2010 with the definitive exposure phase from March 01 to 04, 2010. The aim of the study was the determination of EC₁₀- , EC₂₀ - and EC₅₀ - values of growth rate and yield over a period of 72 h.

Tall oil reaction products with tetraethylenepentamine (CAS no. 68555-22-6) is emulsifiable in water and has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain amido amines/imidazolines 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 bioavaibility and exposure assessment for cationic surface-active substances like the amido amines/imidazolines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavaibility that occurs in the environment with cationic surface-active substances.

The aquatic ecotoxicity tests with amido amines/imidazolines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach 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 to interpret 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. Seven concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 0.010 - 0.032 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Tall oil reaction products with tetraethylenepentamine were analysed at the concentration levels 0.032 and 3.20 mg/L (prepared without algae) and the control at test start via LC-MS/MS analysis. The measured concentrations at test start were in the range of 73 – 111 % of the nominal values. At the end of the test Tall oil reaction products with tetraethylenepentamine was analysed at concentration levels 0.032 and 3.20 mg/L (prepared without algae) and gave recoveries of LOQ - 84 % 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. 146 µg/L could be recovered from the glassware (corresponding to 5 % of 3.2 mg/L). Furthermore imidazolines like the test item have the potential to hydrolyze. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore all effect values are given based on nominal concentrations of the test item. For details of the analytical results please refer to part 9.

Tall oil reaction products with tetraethylenepentamine (CAS No. 68555-22-6) was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₁₀) and yield (E_yC₁₀) after 72 h were 0.395 (0.356 – 0.452) and ≤ 0.010 mg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of specific growth rate (E_rC₅₀) and yield (E_yC₅₀) after 72 h were 0.638 (0.589 – 0.683) and 0.398 (0.330 – 0.683) mg/L, respectively.

After 72 h algae were transferred from the nominal concentrations of 1.00 – 3.20 mg/L and the control to fresh untreated medium and allowed to grow for further 4 – 12 d under test conditions. The test item effect was observed to be reversible at 1.00 mg/L and not reversible at 3.2 mg/L. Therefore, there is potential for recovery following exposure up to 1.00 mg/L (third highest test concentration).

Description of key information

The toxicity to aquatic algae as performed by Noack is a valid algae study and resulted in an 72 hour EC10 and EC50 based on growth of resp. 379.2 and 612.5 µg a.i./L. This was performed for the purpose of performing risk assessment according to the Bulk approach and is considered less suitable for C&L.

For C&L the available

Key value for chemical safety assessment

EC50 for freshwater algae:

612.5 µg/L

EC10 or NOEC for freshwater algae:

379.2 µg/L

Additional information

Two algae toxicity studies have been performed. One study performed to assess the algae toxicity of a range of comparable imidazoline products and one definitive study with Tall oil, reaction products with tetraethylenepentamine (CAS no 68555 -22 -6). The 72 h EC10 based on growth is 395 µg/L related to the nomimal test substance concentration. Corrected for active ingredient which is 96%, the EC10 is reduced to 379.2 µg/L. The test was performed with river water as it is intended to be used in an evaluation of the environmental risks based on the Bulk approach where it is essential to show that the organisms were fully exposed to the nominal test concentration. During the test a decrease of the concentration is observed of about 70%. Imidazolines are susceptible to hydrolysis and the decrease observed could therefore be assigned to either hydrolysis or sorption or both processes. Imidazolines will however only hydrolyze to an amidoamine which is relatively resistant to hydrolysis under ambient conditions. As this hydrolysis will also occur under environmental conditions and amidoamines have a comparable ecotoxicity as the imidazolines the Nominal concentration based on active ingredient is considered as the concentration to be used for the dose effect assesment as no significant adsorption to glassware was observed and the organisms were thus fully exposed the the nominal concentration based on active ingredient. Initially to the imidazoline which gradually changed into an amidoamine during the test. It is considered realistic to relate the observed effects to the nominal values based on active ingredient.

The effects observed in the definitive study are in good agreement with the results observed in the other study.