Pyrolytic lignin

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

other: Analytical method developed for Daphnia and algae medium

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

july 2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

december 2015

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD guidance document No. 23, second edition, GUIDANCE DOCUMENT ON AQUEOUS-PHASE AQUATIC TOXICITY TESTING OF DIFFICULT TEST CHEMI-CALS

Version / remarks:

February 2019

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: SANCO/3029/99: “Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414”

Version / remarks:

rev. 4 dated 11. July 2000

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Low molecular weight organic substances from fast pyrolysis bio-oil

IUPAC Name:

Low molecular weight organic substances from fast pyrolysis bio-oil

Constituent 2

Reference substance name:

Monomers from fast pyrolysis bio-oil

IUPAC Name:

Monomers from fast pyrolysis bio-oil

Constituent 3

Reference substance name:

Dimers from fast pyrolysis bio-oil

IUPAC Name:

Dimers from fast pyrolysis bio-oil

Constituent 4

Reference substance name:

Trimers from fast pyrolysis bio-oil

IUPAC Name:

Trimers from fast pyrolysis bio-oil

Constituent 5

Reference substance name:

Higher oligomers from fast pyrolysis bio-oil

IUPAC Name:

Higher oligomers from fast pyrolysis bio-oil

Constituent 6

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid: viscous

Remarks:

Brown /black highly vicous liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the end of the test, an extra replicate for each treatment and the control without algae was prepared. To avoid adsorption onto the algae cells, the content of the TOC and the content of the two components of the test item in the test solutions was measured in extra replicates without algae cells at the beginning and at the end of the experiment.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Because the test item is a poorly water soluble UVCB substance with constituents of di-ferent water solubility, in accordance with the OECD guidance document no. 23, water-accommodated fractions (WAF) were prepared for the test.
- Loading: WAF was created by weighing the nominal load of 100 / 56 / 32 / 18 / 10 mg/L, adding the corresponding amount of algal medium (demineralised water enriched with minerals but without algae) and shaking vigorously for 24 ± 1 hours. Hence, the real load was 100.3 / 55 / 32.5 / 17.8 / 10.3 mg/L
- Evidence of undissolved material: To remove undissolved components the solutions were filtrated through 0.45 µm PTFE filters
- Other relevant information: As the test item is highly viscous, it was placed in a water bath (30 ± 2 °C) and stirred thoroughly with a stirring staff until it is visibly homogeneous before usage.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): cell concentration of 3 *103 cells/mL
- Method of cultivation: 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
- pre-culture: For the pre-culture an aliquot of the permanent culture was brought into nutrient medium and incubated under continuous lighting for 4 days under test conditions (Lighting: within the specified range of 4440 – 8880 lux; 21– 24 °C). The resulting culture grew exponentially. Before usage, the pre-culture was checked for the absence of cell aggregates and the cell number of the culture was determined.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21.5 – 23.2 °C

pH:

7.6-8.9

Nominal and measured concentrations:

10 / 18 / 32 / 56 / 100 mg/L nominal concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks total volume 65 mL
- Initial cells density: 3 *103 cells/mL
- Control end cells density: 571120 cells/mL
- volume solution: 45 ±1 mL
- No. of vessels per concentration (replicates): 3 test solution+algae, 1 test solution only
- No. of vessels per control (replicates): 6 algae medium+algae, 1 algae medium only

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: under continuous lighting
- Light intensity and quality: within the specified range (4440 – 8880 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Other: appearance of the algae and detect abnormalities

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10, 100 mg/L loading rate
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used: orbital shaker

Reference substance (positive control):

yes

Remarks:

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

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

55.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

62.86 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

34.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

yield

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: concentrations refer to nominal loading rates

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72h ErC50 0.64 mg/L (0.58 – 0.71 mg/L), 72h EyC50 0.32 mg/L (0.31 – 0.33 mg/L)

- Other:

Reported statistics and error estimates:

Calculation of results was performed with the help of validated software. Calculation was performed with Microsoft Excel® and ToxRat® Professional, version 3.3.0.

Any other information on results incl. tables

Non-GLP pre-test results (72h)

Loading ratein mg/L	Inhibition in %
	Growth rate	Yield
Blank control	0	0
1	0.3	1.9
10	1.7	9.7
100	51.5	94.8

Cell Number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
Blank control	Mean	3020	16470	107073	571120
	SD	0	1941	5571	16190
10	Mean	3020	16253	120373	571680
	SD	0	646	7614	9626
18	Mean	3020	15813	125333	544940
	SD	0	603	4490	15522
32	Mean	3020	14900	119687	583733
	SD	0	242	10879	30417
56	Mean	3020	13567	49640	316320
	SD	0	4048	1484	13316
100	Mean	3020	11340	25940	115387
	SD	0	859	1422	1528

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
10	Yes
18	Yes
32	Yes
56	Yes
100	No cells visible

Growth Rate µ, Yield

Nominal Concentration in mg/L	Parameter	Growth Rate (0-72 h) [day^-1]	Yield (0-72 h) [Cell Concentration/mL]
Blank control	Mean	1.75	568100
	SD	0.01	16190
10	Mean	1.75	568660
	SD	0.01	9626
18	Mean	1.73	541920
	SD	0.01	15522
32	Mean	1.75	580713
	SD	0.02	30417
56	Mean	1.55	313300
	SD	0.01	13316
100	Mean	1.21	112367
	SD	0.00	1528

SD =Standard Deviation

Inhibition Values

Nominal concentration in mg/L	% Inhibition
	Growth Rate (0-72 h)	Yield (0-72 h)
Blank control	0	0
10	0,0	-0,1
18	0,9	4,6
32	-0,4	-2,2
56	11,3	44,9
100	30,5	80,2

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

Biological Results of the Test Item

Parameter	Value	95 % confidence interval
NOEL (Growth Rate) 72 h	32 mg/L	--
NOEL (Yield) 72 h	32 mg/L	--
LOEL (Growth Rate) 72 h	56 mg/L	--
LOEL (Yield) 72 h	56 mg/L	--
72h ErL10	55.53 mg/L
72h EyL10	34.13 mg/L	29.41 – 38.00 mg/L
72h ErL50	> 100 mg/L	--
72h EyL50	62.86 mg/L	59.16 – 66.86 mg/L

Analytical Determination, LC-MS-MS

A LC-MS-MS method for the determination of the test item was validated using two components as markers, chosen because they could be clearly assigned as components of the test item and could be quantified in aqueous medium during validation.

At the beginning, the measured concentrations lay between 35 % and 66 % of the loading rates for component 1 and between 14 % and 30 % of the loading rates for component 2. At the end of the test, at the lowest concentration 10 mg/L the measured concentration was lower than LOQ for both components. At the other concentrations the measured concentrations lay between 32 % and 45 % of the loading rates for component 1 and between 9 % and 15 % of the loading rates for component 2.

The details are given in the following tables:

Measured Concentrations and % of Nominal Loading, Component 1

Nominal Concentration in mg/L	Measured Concentration in mg/L  t = 0 h	% of Nominal Loading t = 0 h	Measured Concentration in mg/L t = 72 h	% of Nominal Loading t = 72 h
10	6.61	66%	< LOQ	--
18	9.23	51%	8.11	45%
32	12.75	40%	11.27	35%
56	20.98	37%	18.12	32%
100	34.74	35%	32.61	33%

Measured Concentrations and % of Nominal Loading, Component 2

Nominal Concentration in mg/L	Measured Concentration in mg/L  t = 0 h	% of Nominal Loading t = 0 h	Measured Concentration in mg/L t = 72 h	% of Nominal Loading t = 72 h
10	3.04	30%	< LOQ	--
18	3.99	22%	2.63	15%
32	4.69	15%	3.43	11%
56	8.65	15%	6.39	11%
100	14.01	14%	9.23	9%

Analytical Determination, TOC

Additionally, the total amount of dissolved test item in the test solutions was estimated by calculation based on the carbon content of the test item (60.83 %) and TOC measurement.

Due to the poor solubility of the test item, the measured TOC concentrations were very low. However, at higher loading rates, the measured TOC concentrations also increase.

The almost identical TOC concentrations in the respective test solutions at the beginning and end of the test confirms that the concentration of the test substance or the test substance and its degradation products was present in the test solution over the test period.

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	7.41	7.43	7.77	7.55
10	8.68	8.18	7.51	7.54
18	9.36	8.64	7.50	7.55
32	9.46	9.04	7.59	7.56
56	10.75	10.35	7.75	7.33
100	12.31	11.58	7.35	7.11

Calculated Concentrations TOC

Nominal Concentration Test Item	Calculated TOC (TC-IC)  t = 0 h	Calculated TOC (TC-IC)  t = 72 h
mg/L	mg/L	mg/L
Blank control	0	0
10	1.16	0.65
18	1.87	1.10
32	1.87	1.48
56	3.00	3.02
100	4.96	4.48

Calculated Test item Concentrations

Nominal Concentration Test Item	Calculated Concentration  Test Item  t = 0 h	Calculated Concentration  Test Item  t = 72 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 72 h
mg/L	mg/L	mg/L	%	%
10	1.92	1.07	19%	11%
18	3.07	1.81	17%	10%
32	3.07	2.43	10%	8%
56	4.94	4.96	9%	9%
100	8.15	7.36	8%	7%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

72 h exposure of the green algae, Desmodesmus subspicatus, to the test item resulted in an EL50 growth of >100 mg/l and an EL50 yield of 62.86 mg/L

Executive summary:

One valid experiment was performed exposing for 72 hours the green algae, Desmodesmus subspicatu, to the test item according to OECD 201 guideline. Because the test item is a poorly water soluble UVCB substance with constituents of different water solubility, in accordance with the OECD guidance document no. 23, water-accommodated fractions (WAF) were prepared for the test solution. The study was performed using five loading rates 10 / 18 / 32 / 56 / 100 mg/L.

Analytical monitoring was performed with a LC-MS-MS-Method validated using two components as markers, chosen because they could be clearly assigned as components of the test item and could be quantified in aqueous medium during validation. At the beginning, the measured concentrations lay between 35 % and 66 % of the loading rates for component 1 and between 14 % and 30 % of the loading rates for component 2. At the end of the test, at the lowest loading rate of 10 mg/L, the measured concentration was lower than LOQ for both components. At the other concentrations (loading rates) the measured concentrations lay between 32 % and 45 % of the loading rates for component 1 and between 9 % and 15 % of the loading rates for component 2.

 

Additionally, the total amount of dissolved test item in thetest solutions was estimated by calculation based on the carbon content of the test item (60.83 %) and TOC measurement.Due to the poor solubility of the test item, the correlation between measured concentration (based on TOC measurement) and nominal loading was weak. However, at higher loading rates, the measured TOC concentrations also increase. The almost identical TOC concentrations in the respective test solutions at the beginning and end of the testconfirms that the concentration of the test substance or the test substance and its degradation products was present in the test solution over the test period.

As the test item is a UVCB substance, according to OECD guidance document No. 23 (2019), the results are presented, based on the nominal WAF loading rates (see OECD guidance document no 23, § 177). Growth rate µ and the yield were determined from the cell number.

Significant inhibition of algal growth was observed at the two highest loading rates 56 mg/L and 100 mg/L.

 

The following results for the test item, Pyrolytic Lignin, were determined after 72 hours of exposure:

Endpoint	NOEL	LOEL	EL10	EL50
Growth Rate	32 mg/L	56 mg/L	55.53 mg/L	> 100 mg/L
Yield	32 mg/L	56 mg/L	34.13 mg/L	62.86 mg/L