Tall oil pitch, reaction product with triethylene glycol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-12 to 2017-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented guideline study with GLP and no deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guideline No. 201 for Testing of Chemicals (2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Council Regulation (EC) No. 266/2016 C.3

Deviations:

no

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media were performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture (e.g. UVCB) and is poorly soluble in water and in the permitted solvents.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item: Tall oil pitch, reaction product with triethylene glycol
CAS name: Tall-oil pitch, ester with triethylene glycol
CAS number: 2135769-54-7
Batch number: LABO 16-02
Purity: 100% (UVCB substance)
Water solubility: Sparingly soluble
Appearance: Viscous (liquid), black
Stability under test conditions: Not specified
Expiry date: 2018-11-14

Analytical monitoring:

yes

Details on sampling:

The cell density was measured daily via Chlorophyll a-fluorescence. Microscopic evaluation of the cells at the start and end of the incubation was carried out.

The pH-values at the start of exposure were measured in one additional replicate of each test item loading level and the controls. At the end of exposure, the pH-values were measured from pooled samples of each test item loading and the controls. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure

All loading levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. One additional replicate without algae was incubated under test conditions and analysed after 72 hours. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions. The control replicates were measured at least in triplicates to determine the variability. For the evaluation, the carbon content of the control replicates were subtracted from the carbon content of the test replicates in order to calculate the net carbon content.

Vehicle:

no

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere, is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media was prepared by mixing the test item with dilution water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fraction: Five water accommodated fractions (WAF) were prepared with a nominal loading rate of the test item of 19.8 – 29.6 – 44.4 – 66.7 – 100 mg/L (spacing factor 1.5). The loading concentrations were based on the results of a preliminary range finding test (non-GLP, open system). For each loading level an appropriate amount of the test item was weighed out (not more than ±0.5% of nominal loading). The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with 2 L of dilution water. These dispersions were shaken for 24 hours with at 20 rpm at room temperature. After a separation phase of 1 hour at room temperature, the aqueous phase or WAF’s were removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fractions (WAF) were used in the test.

Test loadings: Per definition of the WAF, all terms related to concentration level will be given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Control Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Reference Item Potassium dichromate was tested as a reference item

Test organisms (species):

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

Details on test organisms:

Test organism: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
Synonyms: Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen, 2016)
Reason for the selectionof the test organism: Pseudokirchneriella subcapitata is a suitable green alga species according to the guideline.
Origin: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen.
Cultivation at test facility: Fresh stocks were prepared every month on Z-Agar and maintained under constant illumination (2567 – 5130 lux, corresponding to 35 - 70 µE ∙ m-2 ∙ s-1).
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22.8 - 23.5 °C

pH:

7.7 - 7.9

Details on test conditions:

Test method: Static procedure
Duration of the test: 72 hours
Replicates: Three replicates per loading level and six for the controls.
Test container and Pre-treatment: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs were used.
Test volume: 100 mL
Dilution water: OECD TG 201 growth medium (according to the guidelines)
Preculture: A three days old preculture,of P. subcapitata prepared in dilution water, was used as inoculum.
Initial cell density: Nominal: approximately 5 x 103 - 104 cells/mL
Actual: 6839 cells/mL
Application: Application was carried out by adding appropriate volumes of the WAFs with algae to the replicate test vessels.
Incubation: The flasks were positioned randomly and repositioned daily.
Temperature (target): Nominal range: 21 - 24°C, controlled at ± 2°C
Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
Light intensity (target): Approximately 4440 to 8880 lux at the approximate level of the test solution, corresponding to 60 to 120 µE*m-2*s-1
Light regime: Continuous illumination
Light homogeneity (target): Within ± 15% from the average light intensity over incubation area

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

44.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

66.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

44.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

66.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

46.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

56.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibtion

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Details on results:

Range finding test
A non-GLP preliminary range finding test under static conditions, over a period of 72 hours was conducted at the test facility with three water accommodated fractions (WAF) of the test item with nominal concentrations of 1.00 – 10.0 – 100 mg/L. Each WAF was prepared separately with dilution water and shaken for 24 hours (20 rpm, room temperature). After a separation phase of 1 hour the WAFs were taken from the approximate centre of the flasks. For all loading levels a Tyndall effect was observed and the media of the highest concentration level was visually turbid throughout the exposure period. In the range finding test, two replicates per concentration and four for the control were tested.

Definitive test
Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the nominal test item loadings.

Table 3. Results of Range Finding Test 0 -72 hours

Nominal loading level of the water accommodated fraction [mg/L]	Growth Rate Inhibition [%]		Yield Inhibition [%]
	Replicate		Replicate
	1	2	1	2
100	6	6	31	28
10.0	-4	-3	-27	-22
1.00	-4	-4	-26	-23

Negative values = growth stimulation

Table 4. Range finding Study TOC content of the Test Media at the Start and End of the exposure

Nominal loading level of the water accommodated fraction [mg/L]	TOC[mg/L]
	Start of exposure (without algae), 0 hours	End of exposure (without algae), 72 hours
100	7.37	4.49
10.0	< LOQ	< LOQ
1.00	< LOQ	< LOQ

Limit of quantification = 2.00 mg/L

Table 5. Definitive Test Algal Cell Densities

Nominal test item loading	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6839	32918	231766	1533131
	2	6839	29273	254976	1606867
	3	6839	27450	211659	1226896
	Mean	6839	29880	232800	1455631
66.7	1	6839	28000	232618	1657805
	2	6839	35564	230605	1808962
	3	6839	28562	244556	1426710
	Mean	6839	30709	235926	1631159
44.4	1	6839	36251	360546	2656417
	2	6839	30510	322192	2101004
	3	6839	35789	279800	2159699
	Mean	6839	34183	320846	2305707
29.6	1	6839	36456	284829	2285687
	2	6839	40500	345515	2379494
	3	6839	34577	408904	2440238
	Mean	6839	37178	346416	2368473
19.8	1	6839	41615	394104	2742503
	2	6839	34993	361368	2799092
	3	6839	31965	300296	2645711
	Mean	6839	36191	351923	2729102
Control	1	6839	39218	319020	2417360
	2	6839	39473	396372	2609305
	3	6839	32886	356988	2728101
	4	6839	28623	223656	2106619
	5	6839	33717	277822	2152381
	6	6839	40465	320939	2245896
	Mean	6839	35730	315800	2376610

Table 6.  Definitive test evaluation of growth rate and yield at 72 hours.

Statistically significant difference of growth rates and yiled compared to control values are marked (+), not significant difference are marked (-). 

Nominal test item loading	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.80	7		1526292	36
	2		1.82	7		1600028	32
	3		1.73	11		1220057	49
	Mean	(+)	1.79	8	(+)	1448792	39
66.7	1		1.83	6		1650966	30
	2		1.86	5		1802123	24
	3		1.78	9		1419871	40
	Mean	(+)	1.82	6	(+)	1624320	31
44.4	1		1.99	-2		2649578	-12
	2		1.91	2		2094165	12
	3		1.92	2		2152860	9
	Mean	(-)	1.94	1	(-)	2298868	3
29.6	1		1.94	1		2278848	4
	2		1.95	0		2372655	0
	3		1.96	-1		2433399	-3
	Mean	(-)	1.95	0	(-)	2361634	0
19.8	1		2.00	-3		2735664	-15
	2		2.00	-3		2792253	-18
	3		1.99	-2		2638872	-11
	Mean	(-)	2.00	-2	(-)	2722263	-15
Control	1		1.96			2410521
	2		1.98			2602466
	3		2.00			2721262
	4		1.91			2099780
	5		1.92			2145542
	6		1.93			2239057
	Mean		1.95			2369771

Table 7:   Definitive test TOC content at the start and end of exposure

Sampling date	2017-09-18 0 hours Start of the exposure		2017-09-21 72 hours End of the exposure
Date of analysis	2017-09-18		2017-10-05
Nominal test item loading [mg/L]	TOC[mg C/L]
	Meas. TOC [mg C/L]	Calc. TOC [mg/L]	Meas. TOC [mg C/L]	Calc. TOC [mg/L]
100	9.54	8.67	3.44	2.65
66.7	8.20	7.33	3.94	3.15
44.4	5.61	4.74	2.43	1.64
29.6	3.88	3.01	2.10	1.31
19.8	< LOQ	< LOQ	3.90	3.11
Control (Rep. 1, 2, 3)	1.21	-	0.814	-
	0.739		0.831
	0.671		0.727
Control (Mean)	0.873	-	0.791	-
Control (±SD)	0.240	-	0.0558	-
LOQ	3.52	-	1.29	-

Table 8. NOEL, LOEL, ELx - values and 95% confidence Intervals of Tall oil pitch, reaction product with triethylene glycol (0 -72 hours) based on nominal test item loading [mg/l]

	Growth Rate Inhibition
NOEL	44.4
LOEL	66.7
ErL10	> 100
ErL20	> 100
ErL50	> 100
	Yield Inhibition
NOEL	44.4
LOEL	66.7
EyL10	46.2 (36.3 – 56.9)
EyL20	56.2 (46.4 – 70.8)
EyL50	> 100

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate inhibition and yield inhibition for Tall oil pitch, reaction product with triethylene glycol was >100 mg/l nominal.

Executive summary:

The toxicity of Tall oil pitch, reaction product with triethylene glycol (Batch no.: LABO 16-02) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD Guideline 201 and Council Regulation (EC) No. 266/2016 C.3. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-values of growth rate and yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Due to the low aqueous solubility and complex nature of the test item the test solutions were prepared as Water Accommodated Fractions (WAF). The EC50 for growth rate inhibition and yield inhibition for Tall oil pitch, reaction product with triethylene glycol was >100 mg/l nominal.

Description of key information

The toxicity of Tall oil pitch, reaction product with triethylene glycol (Batch no.: LABO 16-02) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD Guideline 201 and Council Regulation (EC) No. 266/2016 C.3. The aim of the study was the determination of NOEL, LOEL, EL10, EL20- and EL50-values of growth rate and yield over a period of 72 hours.The study was conducted under static conditions with an initial cell density of 6839 cells/mL. Due to the low aqueous solubility and complex nature of the test item the test solutions were prepared as Water Accommodated Fractions (WAF). The EC50 for growth rate inhibition and yield inhibition for Tall oil pitch, reaction product with triethylene glycol was >100 mg/l nominal.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

44.4 mg/L