Pyrolytic lignin

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 2008

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:

08. Feb. 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. Jul. 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: The content of the TOC and the content of the two components of the test item in the test solutions was measured at the beginning and at the end of the test. The samples at the beginning were taken from the fresh prepared test solutions and from the blank control (M4 medium). At the end of the test, samples were taken from one replicate of the blank control and one replicate of each test solution.

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 different water solubility, in accordance with the OECD guidance document no. 23, water-accommodated fractions (WAF) were prepared for the test.
- Differential loading: to create WAF, the nominal load of 100 / 56 / 32 / 18 / 10 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 ± 1 hours. Real load: 11.3, 18.0, 33.5, 55.3 and 102.3 mg/L. The resulting solutions, the water-soluble fraction, was used as test solutions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: STRAUS, Berlin
- Age at study initiation (mean and range, SD): between 0 and 24 hours
- Method of breeding: multiply by parthenogenesis
- Source: Umweltbundesamt Berlin
- Parental stock:in house breeding since 27. September 2007
- Feeding during test: no

ACCLIMATION
24 hours before the start of the test, the adult animals were separated from the young. 0.67 hours before test start, the adults were caught with the help of a glass tube, and the newborn daphnia (age < 24h) were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20.1 – 20.9 °C

pH:

7.6-7.8

Dissolved oxygen:

8.5-8.8 mg/L O2

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers, nominal volume 50 mL, tall shape
- Volume of solution: 20 ± 5 mL test solution
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 OECD standard medium

OTHER TEST CONDITIONS
- Photoperiod: 16/8 hours, using LED’s

EFFECT PARAMETERS MEASURED: immobilization at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: yes not GLP
- Results used to determine the conditions for the definitive study: yes

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:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: concentrations refer to nominal loading rates

Effect concentrations 2

Key result

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: concentrations refer to nominal loading rates

Results with reference substance (positive control):

- Results with reference substance valid?
- Relevant effect levels: yes
- Dose-response test: yes
- ECx: the values lay within the range of the laboratory (1.20 – 2.82 mg/L)

Any other information on results incl. tables

Non GLP pre-tests (48h)

Loading rate in mg/L		Immobility in %
Blank control		0
1		0
10		0
100		10

 

Immobility

Nominal Concentration in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0	0	0
56	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0

Analytical Determinations, LC-MS-MS

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

 Due to insufficient solubility in the daphnia test medium, neither component 1 nor component 2 was measurable at any time.

The poor solubility is most likely caused by a reaction of the dissolved components with the ingredients of the complexly composed test medium. In the validation study, the solubility was sufficient for determination of the Accuracy. During validation however, the test solutions were prepared by spiking of mediawith test item solutions in acetone and not by preparation of a WAF followed by membrane filtration.

Measured Concentrations at 0 h, Component 1

Nominal Concentration	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean Calculated Concentration
mg/L	counts*min	mg/L	%	--	mg/L	mg/L
QC sample (1.1 mg/L)	30767	0.9485	86.2%	--	1.10	--
	31040.2	0.9571	87.0%	--	1.10
Blank control	0	-0.0120	90.5%	--	< LOQ	--
	0	-0.0120	90.5%	-	< LOQ
10	432.5	0.0015	90.5%	10	< LOQ	< LOQ
	440.0	0.0017	90.5%	10	< LOQ
18	0.0	-0.0120	90.5%	20	< LOQ	< LOQ
	445.9	0.0019	90.5%	20	< LOQ
32	282.0	-0.0032	90.5%	20	< LOQ	< LOQ
	285.0	-0.0031	90.5%	20	< LOQ
56	248.9	-0.0043	90.5%	40	< LOQ	< LOQ
	453.0	0.0021	90.5%	40	< LOQ
100	363.6	-0.0007	90.5%	100	< LOQ	< LOQ
	290.8	-0.0030	90.5%	100[TV2]	< LOQ
QC sample (1.1 mg/L)	33274.7	1.0268	93.3%	--	1.10	--
	34046.6	1.0509	95.5%	--	1.10

LOQ = 0.1 mg/L

Mean recovery of the QC sampleof 90.5 % was taken into account

Measured Concentrations at 48 h, Component 1

Nominal Concentration	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean Calculated Concentration
mg/L	counts*min	mg/L	%	--	mg/L	mg/L
QC sample (1.1 mg/L)	34433,4	1,0630	96,6%	--	1.10	--
	34209,3	1,0560	96,0%	--	1.10
Blank control	0,0	-0,0120	--	--	< LOQ	--
	0,0	-0,0120	--	--	< LOQ
10	0,0	-0,0120	--	10	< LOQ	< LOQ
	233,6	-0,0047	--	10	< LOQ
18	32,9	-0,0110	--	20	< LOQ	< LOQ
	0,0	-0,0120	--	20	< LOQ
32	0,0	-0,0120	--	20	< LOQ	< LOQ
	80,9	-0,0095	--	20	< LOQ
56	0,0	-0,0120	--	40	< LOQ	< LOQ
	57,8	-0,0102	--	40	< LOQ
100	0,0	-0,0120	--	100	< LOQ	< LOQ
	165,8	-0,0069	--	100	< LOQ
QC sample (1.1 mg/L)	35749,1	1,1041	100,4%	--	1.10	--
	36889,6	1,1397	103,6%	--	1.10

LOQ = 0.1 mg/L

Mean recovery of the QC sample of 99.2 % was not taken into account

Measured Concentrations at 0 h, Component 2

Nominal Concentration	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean Calculated Concentration
mg/L	counts*min	mg/L	%	--	mg/L	mg/L
QC sample (1.1 mg/L)	84828.4*	0.7702	70.0%*	--	1.10	--
	110023	0.9929	90.3%	--	1.10
Blank control	0	0.0205	92.3%	--	< LOQ	--
	0	0.0205	92.3%	-	< LOQ
10	669.5	0.0264	92.3%	10	< LOQ	< LOQ
	744.5	0.0271	92.3%	10	< LOQ
18	678.1	0.0265	92.3%	20	< LOQ	< LOQ
	763.7	0.0272	92.3%	20	< LOQ
32	1008.5	0.0294	92.3%	20	< LOQ	< LOQ
	1014.8	0.0294	92.3%	20	< LOQ
56	603.4	0.0258	92.3%	40	< LOQ	< LOQ
	702.2	0.0267	92.3%	40	< LOQ
100	449.6	0.0244	92.3%	100	< LOQ	< LOQ
	439.1	0.0244	92.3%	100	< LOQ
QC sample (1.1 mg/L)	116081.7	1.0464	95.1%	--	1.10	--
	111618.7	1.0070	91.5%	--	1.10

LOQ = 0.1 mg/L

*first QC not taken into account because measurement not yet sufficiently equilibrated

Mean recovery of the other 3 QC sample of 92.3 % was taken into account

Measured Concentrations at 48 h, Component 2

Nominal Concentration	Area	Measured Concentration	QC recovery	Dilution factor	Calculated Concentration	Mean Calculated Concentration
mg/L	counts*min	mg/L	%	--	mg/L	mg/L
QC sample (1.1 mg/L)	123361.4	1.1108	101.0%	--	1.10	--
	123478.4	1.1118	101.1%	--	1.10
Blank control	0.0	0.0205		--	< LOQ	--
	0.0	0.0205		-	< LOQ
10	111.9	0.0215		10	< LOQ	< LOQ
	77.4	0.0212		10	< LOQ
18	146.7	0.0218		20	< LOQ	< LOQ
	0.0	0.0205		20	< LOQ
32	51.0	0.0209		20	< LOQ	< LOQ
	0.0	0.0205		20	< LOQ
56	105.7	0.0214		40	< LOQ	< LOQ
	0.0	0.0205		40	< LOQ
100	0.0	0.0205		100	< LOQ	< LOQ
	94.8	0.0213		100	< LOQ
QC sample (1.1 mg/L)	128883.4	1.1596	105.4%	--	1.10	--
	126726.4	1.1405	103.7%	--	1.10

LOQ = 0.1 mg/L

Mean recovery of the QC sample of 102.8 % was taken into account

 

Analytical Determinations, TOC

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 concentration were very low. TOC measurement was not specific and at low concentrations (< 1 mg/L) not very exact because the test medium with daphnia also contained TOC at low concentration. However, the measured TOC concentrations in the three highest concentrations at the beginning and at the end were clearly higher than the TOC in the control. And it was clearly recognisable that at higher loading rates, the measured TOC concentrations also increase.

Although the two components could not be quantified with the LC-MS-MS measurement, the TOC measurement does show that a part of the test substance was dissolved in the test solutions.

The almost identical TOC concentrations in the respective test solutions at the beginning and end of the test confirm 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 = 48 h	Measured IC  t = 0 h	Measured IC  t = 48 h
mg/L	mg/L	mg/L	mg/L	mg/L
Blank control	10.17	10.92	10.04	10.73
10	10.68	11.38	10.03	10.96
18	11.49	11.83	10.25	11.13
32	11.92	12.48	10.48	10.64
56	12.61	12.76	10.33	10.81
100	13.22	13.07	10.42	10.58

Calculated Concentrations TOC

Nominal Concentration Test Item	Calculated TOC (TC-IC)  t = 0 h	Calculated TOC (TC-IC)  t = 48 h
mg/L	mg/L	mg/L
Blank control	0.13	0.19
10	0.64	0.42
18	1.24	0.70
32	1.44	1.84
56	2.27	1.95
100	2.80	2.49

Calculated Test item Concentrations

Nominal Concentration Test Item	Calculated Concentration  Test Item  t = 0 h	Calculated Concentration  Test Item  t = 48 h	% of Nominal concentration t = 0 h	% of Nominal concentration t = 48 h
mg/L	mg/L	mg/L	%	%
10	1.06	0.69	11%	7%
18	2.04	1.16	11%	6%
32	2.37	3.02	7%	9%
56	3.74	3.20	7%	6%
100	4.60	4.10	5%	4%

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

48 h exposure of daphnia magna to the test item resulted in a EC50 >100 mg/L

Executive summary:

One valid experiment was performed according to the OECD guideline 202. Testing solution was prepared following OECD guidance document no. 23, water-accommodated fractions (WAF) due to the poor solubility in water of the test item being a UVCB substance with constituents of different water solubility. The study was performed using five loading rates 10 / 18 / 32 / 56 / 100 mg/L.

Analytical monitoring was performed, at the start and at the end of the test, 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.

Due to insufficient solubility in the daphnia test medium, neither component 1 nor component 2 was measurable at any time. However, during validation, preparing the test solution by spiking of media with test item solutions in acetone, the solubility was sufficient for determination of the Accuracy.

 

Additionally, the total amount of dissolved test item in the 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 correlation between measured concentration (based on TOC measurement) and nominal loading was very weak. However, at higher loading rates, the measured TOC concentrations also very slightly increase.

The almost identical TOC concentrations in the respective test solutions at the beginning and end of the test confirm 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). No immobilization was observed after 24 and 48 h in animals exposed to either test solution or blanck control.

The following results were determined for the test item, Pyrolytic Lignin (species: Daphnia magna):

24h-EL₅₀> 100 mg/L
48h-EL₅₀> 100 mg/L