Pyrolytic sugar

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:

1,6-anhydro-β-D-glucose

EC Number:

207-855-0

EC Name:

1,6-anhydro-β-D-glucose

Cas Number:

498-07-7

Molecular formula:

C6H10O5

IUPAC Name:

(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol

Constituent 2

Reference substance name:

Glycollaldehyde

EC Number:

205-484-9

EC Name:

Glycollaldehyde

Cas Number:

141-46-8

Molecular formula:

C2H4O2

IUPAC Name:

2-hydroxyacetaldehyde

Constituent 3

Reference substance name:

Organic acids

IUPAC Name:

Organic acids

Constituent 4

Reference substance name:

Ketones

IUPAC Name:

Ketones

Constituent 5

Reference substance name:

Phenols

IUPAC Name:

Phenols

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

Constituent 7

Reference substance name:

Oligomers of sugars and anhydrosugars

IUPAC Name:

Oligomers of sugars and anhydrosugars

Test material form:

liquid: viscous

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: 10, 18.0, 33, 55.8 and 100.5 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:

7.4-7.9

Dissolved oxygen:

8.3-9 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.:

81.05 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.:

83.8 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: t he values lay within the range of the laboratory 2.22 mg/L

Reported statistics and error estimates:

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.3.0.

Any other information on results incl. tables

Non GLP pre-test (48h)

Loading rate in mg/L	Immobility in %
Blank control	0
1	0
10	0
100	70

Immobility

Nominal Concentration in mg/L	Immobility 24 hours					Immobility 48 hours
	absolute				in %	absolute				in %
Blank control	1	0	0	0	5	1	0	0	0	5
10	0	0	0	0	0	0	0	0	0	0
18	1	0	0	0	5	1	0	1	0	10
32	0	0	0	0	0	0	0	0	0	0
56	0	0	0	0	0	0	0	0	0	0
100	3	5	4	4	80	4	5	4	4	85

Analytical Determinations, LC-MS-MS

A LC-MS-MS-Method for the determination of Pyrolytic Sugar 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. Due to insufficient solubility in the daphnia test medium, at the beginning of the test component 1 was measurable only at the two highest loading rates 56 mg/L and 100 mg/L and at the end of the test only at the highest loading rate 100 mg/L.

Component 2 was measurable at the beginning only at the three highest loading rates 32 mg/L, 56 mg/L and 100 mg/L and at the end of the test at the two highest loading rates 56 mg/L and 100 mg/L.

For component 1 the measured concentrations lay between 75 % and 90 % of the loading rates at the beginning, and between 0 % and 85 % at the end of the study. For component 2 the measured concentrations lay between 39 % and 90 % of the loading rates at the beginning, and between 0 % and 30 % at the end of the study.

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 = 48 h	% of Nominal Loading t = 48 h
56	42.18	75%	< LOQ	--
100	90.10	90%	85.43	85%

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 = 48 h	% of Nominal Loading t = 48 h
32	12.42	39%	< LOQ	--
56	47.83	85%	16.64	30%
100	89.92	90%	80.43	80%

Analytical Determinations, 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 (48.73 %) and TOC measure-ment. The test item concentration based on TOC measurement lay between 84 % and 113 % of the loading rates at the beginning of the test, and between 79 % and 98 % at the end of the test.

The almost identical measured TOC concentrations and measured concentrations of the both markers 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 were present in the test solutions 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	14.69	11.04	10.06	10.34
10	20.08	14.66	9.97	10.74
18	22.92	18.63	10.12	10.98
32	28.64	22.95	9.67	10.66
56	37.06	34.46	9.57	10.84
100	58.91	57.58	8.89	10.17

Calculated Concentrations TOC

Nominal Concentration Test Item	Calculated TOC (TC-IC)  t = 0 h	Corrected TOC t = 0 h*	Calculated TOC (TC-IC)  t = 48 h
mg/L	mg/L		mg/L
Blank control	4.62	--	0.68
10	10.11	5.49	3.92
18	12.8	8.18	7.64
32	18.98	14.36	12.29
56	27.49	22.87	23.61
100	50.02	45.4	47.61

* The TOC of the blank control was subtracted from the TOC of the test solutions at the measurement on day 0

 

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	11.27	8.04	113%	80%
18	16.79	15.68	93%	87%
32	29.47	25.22	92%	79%
56	46.93	48.45	84%	87%
100	93.17	97.70	93%	98%

Within this study, 2 selected components of the UVCB were analytically verified. This to evaluate availability and compositional stability of the UVCB within the WAF. In addition, TOC was also monitored with the same purpose. 

This approach allowed confirmation of the presence of the substance in solution. However, due to variability of results and difficulty to link concentrations of components and/or TOC to overall nominal levels, the concentrations are presented as loading rates and not as re-calculated concentrations. This is in line with OECD Guideline 23 (difficult to test substances).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

48 h exposure of daphnia magna to Pyrolytic Sugar resulted in a EL50 of 81.05 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.

Due to insufficient solubility in the daphnia test medium, at the beginning of the test component 1 was measurable only at the two highest loading rates 56 mg/L and 100 mg/L and at the end of the test only at the highest loading rate 100 mg/L.

Component 2 was measurable at the beginning only at the three highest loading rates 32 mg/L, 56 mg/L and 100 mg/L and at the end of the test at the two highest loading rates 56 mg/L and 100 mg/L.

For component 1the measured concentrations lay between 75 % and 90 % of the loading rates at the beginning and between 0 % and 85 % at the end of the study. For component 2 the measured concentrations lay between 39 % and 90 % of the loading rates at the beginning between 0 % and 30 % at the end of the study.

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 (48.73 %) and TOC measurement. The test item concentration based on TOC measurement lay between 84 % and 113 % of the loading rates at the beginning of the test, and between 79 % and 98 % at the end of the test.

The almost identical measured TOC concentrations and measured concentrations of both markers 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 soultion 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). Significant immobility was observed only at the highest loading rate of 100 mg/L. In the blank control one animal (5 %) was immobilised which can be stated as not significant. The concentration-response curve after 48 hours was extremely steep, showing a 0 to 85 % rise in immobilisation between two successive treatments. The following results were determined for the test item, Pyrolytic Sugar (species: Daphnia magna):

24h-EL₅₀= 83.8 mg/L
48h-EL₅₀= 81.05 mg/L