Glycerides, tall-oil mono-

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:

09. Apr. 2018 to 13. Apr. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBI-TION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The content of DOC in the test vessels was measured at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing 99.75 mg/L of the test item with the corresponding amount of algal medium (de-mineralised water enriched with minerals but without algae) and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. The lower treat-ments were prepared by dilution of this WAF with nutrient medium. This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at very low loading rates. In a non-GLP pre-test at the loading rate 1 mg/L clear inhi-bition of algal growth was observed.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification
Unicellular: freshwater green alga.
Genus: Desmodesmus
Species: subspicatus SAG Strain Number 86.81
Taxonomic position: Chlorophyta - Chlorophyceae

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sci-encebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen). The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not specified

Hardness:

Not specified

Test temperature:

21.8 – 23.5 °C

pH:

7.5 - 7.9

Dissolved oxygen:

Not specified

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

1, 3.2, 10, 32, 100 mg/L nominal concentration
The concentrations to be tested based on non GLP pre-tests

Details on test conditions:

Pre-Culture
Four days before the start of each test, an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 96 hours under test con-ditions (5000 lux, 21.7 – 23.1°C). The resulting culture grew exponentially.
Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of culture was determined.

Performance of the Study
For each treatment, 200 mL of the respective test item solution was mixed with the neces-sary amount of algal pre-culture (0.363 mL) to achieve a cell concentration of 2.0*103 cells/mL. For the blank control, 350 mL nutrient medium was used instead of test item so-lution and mixed with the necessary amount of algal pre-culture (0.635 mL).
The real cell concentration at the beginning of the test was measured with an electronic particle counter in the blank control solution. This measured value was used as start cell concentration for all replicates.
From these mixtures samples for the analytical determination were taken, afterwards the pH-value was measured.
The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension. Before the start of incubation and every 24 ± 1 hours, the cell number was determined with an electronic particle counter. After the test, the pH value in treatments and blank control was measured again.
At the end of the test, the treatments were examined microscopically in order to assess the appearance of the algae and detect abnormalities (e.g. caused by the exposure to the test item).
The content of DOC in the test vessels was measured at the start and at the end of the test.

Experimental Conditions
Treatments tested: 1 / 3.2 / 10 / 32 / 100 mg/L nominal concentration. The concentrations to be tested based on non GLP pre-tests
Number of replicates: 6 replicates for the blank control 3 replicates for each treatment
Vessels: glass flasks total volume 65 mL
Duration: 72 hours
Lighting: 5000 lux
Temperature: 21.8 – 23.5 °C
Replicates (Blank control): 45 ±1 mL algal medium and algae
Replicates (Treatments): 45 ±1 mL test solution and algae

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate & yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growht rate & yield

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

DISCUSSION
One valid experiment was performed.
The study was performed using 5 concentrations ranging from 1 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation times.
Significant inhibition of algal growth was observed at the following concentrations:
10 – 100 mg/L
At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solu-tions using a carbon analyser.
The test item could only be detected in the highest concentration. All other measured values are approximately in the same range as the blank control. However, due to the clear dose-response relationship, it is clear that dissolved test item was present in solution.
Therefore, the statistical evaluation was carried out with the nominal concentrations.

Results with reference substance (positive control):

72h ErC50: 0.90 mg/L
72h EyC50: 0.42 mg/L

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software (Microsoft Ex-cel®). The estimation of the biological data was accomplished using the software ToxRat® Professional, version 3.2.1.

Biological Results of K₂Cr₂O₇

Parameter	Value	95% confidence interval
72h ErC50	0.90 mg/L	0.77 – 1.1 mg/L
72h EyC50	0.42 mg/L	0.35 – 0.51 mg/L

 

Cell Numbers

The cell numbers were determined with an electronic particle counter. The means and standard deviations of the cell numbers of the blank control and the treatments are presented in the following table:

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	2000	10333	61580	294913
Blank control	SD	0	1244	8677	21154
1	Mean	2000	9927	48080	308387
1	SD	0	794	8254	13710
3.2	Mean	2000	8213	34447	279313
3.2	SD	0	1435	2311	45673
10	Mean	2000	8520	42673	246220
10	SD	0	225	2706	22986
32	Mean	2000	9433	30933	197673
32	SD	0	1417	1363	26872
100	Mean	2000	5633	21553	59227
100	SD	0	376	1389	8584

SD = Standard Deviation

 

Light Intensity

In the following table, the light intensity during the test is stated:

Light Intensity

	0 h	24 h	48 h	72 h
Light intensity [Lux]	5000	5000	5000	5000

 

pH values

In the following table, the pH values measured at the start and end of the test are stated:

pH values

Nominal Concentration in mg/L	0 h	72 h
Blank control	7.6	7.7
1	7.7	7.9
3.2	7.6	7.8
10	7.6	7.6
32	7.7	7.5
100	7.6	7.5

 

Microscopical Observations

In the following table, the appearance of the algae at the end of the test is stated:

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
1	Yes
3.2	Yes
10	Yes
32	Yes
100	Yes

 

Analytical Determination

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

The test item could only be detected in the highest concentration. All other measured values are approximately in the same range as the blank control. However, due to the clear dose-response relationship, it is clear that dissolved test item was present in solution.

Therefore, the statistical evaluation was carried out with the nominal concentrations.

The details are given in the following tables:

Measured Concentrations IC and TC

Nominal Concentration Test Item	Measured TC t = 0 h	Measured TC t = 72 h	Measured IC t = 0 h	Measured IC t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	7.30	7.00	6.84	6.21
1	7.44	6.64	7.01	6.08
3.2	7.64	6.58	7.00	6.09
10	8.24	6.34	6.87	5.77
32	9.89	6.75	7.05	5.74
100	15.78	7.38	6.71	5.87

LOQ (Limit of quantification) TC = 5.43 mg/L

LOQ (Limit of quantification) IC = 2.03 mg/L

 

Measured Concentrations DOC

Nominal Concentration Test Item	Measured DOC (TC-IC) t = 0 h	Measured DOC (TC-IC) t = 72 h	Measured DOC minus blank control t = 0 h	Measured DOC minus blank control t = 72 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	0.46	0.79	--	--
1	0.44	0.55	-0.03	-0.23
3.2	0.64	0.49	0.18	-0.29
10	1.37	0.57	0.91	-0.22
32	2.84	1.01	2.38	0.22
100	9.06	1.51	8.60	0.72

 

Calculated Test Item Concentrations

Nominal Concentration Test Item	Calculated Concentration t = 0 h	Calculated Concentration t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	--	--	--	--
1	0.0	-0.3	-4	-32
3.2	0.3	-0.4	8	-13
10	1.3	-0.3	13	-3
32	3.3	0.3	10	1
100	12.1	1.0	12	1

 

Growth Rate, Yield

From the cell numbers, the Growth Rate μ and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Growth Rate μ, Yield

Nominal Concentration in mg/L	Parameter	Growth rate (0-72h) [day^-1]	Yield (0-72h) [Cell Concentration/mL]
Blank control	Mean	1.66	292913
	SD	0.02	21154
1	Mean	1.68	306387
	SD	0.01	13710
3.2	Mean	1.64	277313
	SD	0.06	45673
10	Mean	1.60	244220
	SD	0.03	22986
32	Mean	1.53	195673
	SD	0.04	26872
100	Mean	1.13	57227
	SD	0.05	8584

SD = Standard Deviation

 

Inhibition

The following mean inhibition values were calculated for the Growth Rate μ and the Yield.

Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72h)	Yield (0-72h)
Blank control	0	0
1	-0.9	-4.6
3.2	1.2	5.3
10	3.6	16.6
32	8.1	33.2
100	32.3	80.5

Negative inhibition values indicate a stimulation of alga growth compared to the blank control.

 

Measured Cell Numbers

The measured cell numbers for each replicate are presented in the following table:

Cell numbers measured

Nominal concentration in mg/L	Cell Number/mL
	0 h	24 h	48 h	72 h
Blank control	100	447	3641	13372
	100	524	2713	13672
	100	573	2722	14548
	100	445	3601	15599
	100	595	3013	15340
	100	516	2784	15943
1	100	542	2830	16105
	100	477	2376	15419
	100	470	2006	14734
3.2	100	491	1718	15550
	100	353	1609	14999
	100	388	1840	11348
10	100	433	1979	10984
	100	432	2230	12962
	100	413	2192	12987
32	100	453	1501	9126
	100	550	1514	9090
	100	412	1625	11435
100	100	291	1087	2933
	100	260	1004	2547
	100	294	1142	3404

 

Calculated Cell Numbers

The cell number in the solution were calculated (dilution factor 20). The single values and the corresponding means and standard deviations are presented in the following table:

Cells Numbers calculated

Nominal Conc. in mg/L	Replicate	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	1	2000	8940	72820	267440
	2	2000	10480	54260	273440
	3	2000	11460	54440	290960
	4	2000	8900	72020	311980
	5	2000	11900	60260	306800
	6	2000	10320	55680	318860
	Mean	2000	10333	61580	294913
	SD	0	1244	8677	21154
1	1	2000	10840	56600	322100
	2	2000	9540	47520	308380
	3	2000	9400	40120	294680
	Mean	2000	9927	48080	308387
	SD	0	794	8254	13710
3.2	1	2000	9820	34360	311000
	2	2000	7060	32180	299980
	3	2000	7760	36800	226960
	Mean	2000	8213	34447	279313
	SD	0	1435	2311	45673
10	1	2000	8660	39580	219680
	2	2000	8640	44600	259240
	3	2000	8260	43840	259740
	Mean	2000	8520	42673	246220
	SD	0	225	2706	22986
32	1	2000	9060	30020	182520
	2	2000	11000	30280	181800
	3	2000	8240	32500	228700
	Mean	2000	9433	30933	197673
	SD	0	1417	1363	26872
100	1	2000	5820	21740	58660
	2	2000	5200	20080	50940
	3	2000	5880	22840	68080
	Mean	2000	5633	21553	59227
	SD	0	376	1389	8584

SD = Standard Deviation

 

Growth Rate μ

The growth rate μ for each replicate and the corresponding means and standard deviation are given in the following table:

Growth Rate μ

Nominal Conc. in mg/L	Replicate	Growth Rate μ [day^-1]
		24 h	48 h	72 h
Blank control	1	1.50	1.80	1.63
	2	1.66	1.65	1.64
	3	1.75	1.65	1.66
	4	1.49	1.79	1.68
	5	1.78	1.70	1.68
	6	1.64	1.66	1.69
	Mean	1.64	1.71	1.66
	SD	0.12	0.07	0.02
1	1	1.69	1.67	1.69
	2	1.56	1.58	1.68
	3	1.55	1.50	1.66
	Mean	1.60	1.58	1.68
	SD	0.08	0.09	0.01
3.2	1	1.59	1.42	1.68
	2	1.26	1.39	1.67
	3	1.36	1.46	1.58
	Mean	1.40	1.42	1.64
	SD	0.17	0.03	0.06
10	1	1.47	1.49	1.57
	2	1.46	1.55	1.62
	3	1.42	1.54	1.62
	Mean	1.45	1.53	1.60
	SD	0.03	0.03	0.03
32	1	1.51	1.35	1.50
	2	1.70	1.36	1.50
	3	1.42	1.39	1.58
	Mean	1.54	1.37	1.53
	SD	0.15	0.02	0.04
100	1	1.07	1.19	1.13
	2	0.96	1.15	1.08
	3	1.08	1.22	1.18
	Mean	1.03	1.19	1.13
	SD	0.07	0.03	0.05

SD = Standard Deviation

 

Yield

The Yield for each replicate and the corresponding means and standard deviations are given in the following table:

Yield

Nominal Conc. in mg/L	Replicate	Yield [Cell Concentration/mL]
		72 h
Blank control	1	265440
	2	271440
	3	288960
	4	309980
	5	304800
	6	316860
	Mean	292913
	SD	21154
1	1	320100
	2	306380
	3	292680
	Mean	306387
	SD	13710
3.2	1	309000
	2	297980
	3	224960
	Mean	277313
	SD	45673
10	1	217680
	2	257240
	3	257740
	Mean	244220
	SD	22986
32	1	180520
	2	179800
	3	226700
	Mean	195673
	SD	26872
100	1	56660
	2	58940
	3	66080
	Mean	67227
	SD	8584

SD = Standard Deviation

 

Inhibition Values of Single Replicates

The inhibition values at the end of the test of the single replicates are presented in the following table:

Inhibition Values at the end of the test (72 hours)

Nominal Conc. in mg/L	Replicate	Inhibition Growth Rate μ 72 hours	Inhibition Yield 72 hours
Blank control	1	1.92	9.38
	2	1.47	7.33
	3	0.23	1.35
	4	-1.17	-5.83
	5	-0.84	-4.06
	6	-1.61	-8.18
	Mean	0	0
	SD	1.45	7.22
1	1	-1.81	-9.28
	2	-0.94	-4.60
	3	-0.03	0.08
	Mean	-0.93	-4.60
	SD	0.89	4.68
3.2	1	-1.11	-5.49
	2	-0.38	-1.73
	3	5.20	23.20
	Mean	1.24	5.33
	SD	3.45	15.59
10	1	5.86	25.68
	2	2.54	12.18
	3	2.50	12.01
	Mean	3.63	16.62
	SD	1.93	7.85
32	1	9.57	38.37
	2	9.65	38.62
	3	5.05	22.61
	Mean	8.09	33.20
	SD	2.63	9.17
100	1	32.31	80.66
	2	35.14	83.29
	3	29.33	77.44
	Mean	32.26	80.46
	SD	2.91	2.93

SD = Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The following results for the test item LUMULSE GMT-K were determined:

NOEC LOEC EC10 EC50
Growth Rate 3.2 mg/L 10 mg/L 35.79 mg/L > 100 mg/L
Yield 3.2 mg/L 10 mg/L 9.76 mg/L 43.60 mg/L

Executive summary:

Determination of the toxicity of LUMULSE GMT-K against Desmodesmus subspicatus according to OECD 201 resp. EU C.3

 

Findings and Results:

One valid experiment was performed.

The study was performed using 5 concentrations ranging from 1 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations:

10 – 100 mg/L

 

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solu-tions using a carbon analyser.

The test item could only be detected in the highest concentration. All other measured values are approximately in the same range as the blank control. However, due to the clear dose-response relationship, it is clear that dissolved test item was present in solution.

Therefore, the statistical evaluation was carried out with the nominal concentrations (cf. OECD Guideline No. 201, paragraph 40, page 6).

Since the test item is an UVCB, the EC50 value (growth rate) calculated by ToxRat® by extrapolation is not given but a range.

 

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

 

The following results for the test item LUMULSE GMT-K were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	3.2 mg/L	10 mg/L	35.79 mg/L	> 100 mg/L
Yield	3.2 mg/L	10 mg/L	9.76 mg/L	43.60 mg/L

 

Description of key information

The following results for the test item LUMULSE GMT-K were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	3.2 mg/L	10 mg/L	35.79 mg/L	> 100 mg/L
Yield	3.2 mg/L	10 mg/L	9.76 mg/L	43.60 mg/L

 

Key value for chemical safety assessment

EC50 for freshwater algae:

43.6 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

The study was performed using 5 concentrations ranging from 1 to 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate μ and the yield1 were determined from the cell number at the respective observation times.

Significant inhibition of algal growth was observed at the following concentrations: 10 – 100 mg/L

At the start and at the end of the test, the content of the test item in the test solutions was estimated by determination of the dissolved organic carbon (DOC) content in the test solutions using a carbon analyser.

The test item could only be detected in the highest concentration. All other measured values are approximately in the same range as the blank control. However, due to the clear dose-response relationship, it is clear that dissolved test item was present in solution.

Therefore, the statistical evaluation was carried out with the nominal concentrations.

Since the test item is an UVCB, the EC50 value (growth rate) calculated by ToxRat® by extrapolation is not given but a range.

 

The following results for the test item LUMULSE GMT-K were determined:

Results of the test item

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	3.2 mg/L	10 mg/L	35.79 mg/L	> 100 mg/L
Yield	3.2 mg/L	10 mg/L	9.76 mg/L	43.60 mg/L