Pyrolytic sugar

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:

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: At the end of the test, an extra replicate for each treatment and the control without algae was prepared for analytical verification purpose. 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 the 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 different 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 / 32.8 / 17.8 / 10 mg/L
- Evidence of undissolved material: To remove undissolved compo-nents 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:

7.3-7.9

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): 3test 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
- 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.:

23.96 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.:

7.96 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.:

< 10 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.:

11.96 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.:

7.77 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.:

< 10 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.:

<= 10 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.:

<= 10 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)

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

Not-GLP pre-test results (72h)

Nominal Concentration in mg/L	Inhibition in %
	Growth rate	Yield
Blank control	0	0
1	0.8	4.7
10	2.9	15.4
100	60.6	97.0

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	12273	63193	391460
	SD	0	876	8181	12590
18	Mean	3020	8520	16447	48660
	SD	0	1507	1052	2520
32	Mean	3020	7467	14153	27487
	SD	0	429	866	853
56	Mean	3020	8020	17487	18580
	SD	0	439	760	352
100	Mean	3020	14980	15380	14980
	SD	0	886	661	629

Microscopical Observations

Nominal Concentration in mg/L	Normal and Healthy Appearance of the Algae
Blank control	Yes
10	Yes
18	No cells visible
32	No cells visible
56	No cells visible
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	1.62	388440
	SD	0.01	12590
18	Mean	0.93	45640
	SD	0.02	2520
32	Mean	0.74	24467
	SD	0.01	853
56	Mean	0.61	15560
	SD	0.01	352
100	Mean	0.53	11960
	SD	0.01	629

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	7.2	31.6
18	47.0	92.0
32	57.9	95.7
56	65.3	97.3
100	69.5	97.9

Biological Results of the Test Item

Parameter	Value	95 % confidence interval
NOEL (Growth Rate) 72 h	< 10 mg/L	--
NOEL (Yield) 72 h	< 10 mg/L	--
LOEL (Growth Rate) 72 h	≤ 10 mg/L	--
LOEL (Yield) 72 h	≤ 10 mg/L	--
72h ErL10	7.96 mg/L	5.26 – 12.02 mg/L
72h EyL10	7.77 mg/L*	7.27 – 8.30 mg/L
72h ErL;50	23.96 mg/L	14.52 – 38.79 mg/L
72h EyL50	11.62 mg/L	10.74 – 12.53 mg/L

Analytical Determination, LC-MS-MS

A LC-MS-MS-Method for the determination of Pyrolytic Sugar was validated using two components as markers. At the beginning, the measured concentrations lay between 98 % and 106 % of the loading rates for component 1 and between 95 % and 100 % of the loading rates for component 2. At the end of the test, most likely due to an incorrect sampling which cannot be explained afterwards, the measured concentrations for both components at the concentration 18 mg/L and 32 mg/L were not plausible.

However, the treatments were prepared correctly. This was confirmed by the measured start concentrations of both components and the plausible measured TOC concentration at the beginning and at the end of the test as well as the plausible inhibition values in the algae test

In the other treatments the measured concentrations at the end of the test lay between 95 % and 119 % of the loading rates for component 1 and between 72 % and 79 % 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	10.56	106%	9.53	95%
18	17.86	99%	< LOQ*	0%*
32	33.42	104%	11.65*	36%*
56	57.46	103%	59.37	106%
100	98.03	98%	119.44	119%

* implausible values, very likely caused by an error during sampling

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	9.70	97%	7.20	72%
18	17.59	98%	0.26	1%*
32	31.98	100%	13.39	42%*
56	53.35	95%	44.45	79%
100	99.59	100%	79.29	79%

* implausible values, very likely caused by an error during sampling

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 (48.73 %) and TOC measurement. The test item concentration based on TOC measurement lay between 91 % and 101 % of the loading rates at the beginning of the test, and between 74 % and 98 % at the end of the test.

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 sub-stance and its degradation products was stable 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	12.42	11.84	7.51	7.44
18	15.49	14.05	7.51	7.55
32	21.91	20.30	7.33	6.96
56	31.75	30.12	6.77	5.91
100	52.85	52.65	6.78	4.75

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	4,90	4,40
18	7,98	6,50
32	14,58	13,34
56	24,98	24,20
100	46,07	47,90

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	10.06	9.03	101%	90%
18	16.37	13.34	91%	74%
32	29.91	27.38	93%	86%
56	51.26	49.67	92%	89%
100	94.54	98.29	95%	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:

72 h exposure of the green algae, Desmodesmus subspicatus, to Pyrolytic Sugar resulted in an EL50 growth of 23.96 mg/l and an EL50 yield of 11.62 mg/L

Executive summary:

One valid experiment was performed exposing for 72 hours the green algae, Desmodesmus subspicatu, to Pyrolytic Sugar according to OECD 201 guideline. Because the test item is a poorly 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 monitring 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 98 % and 106 % of the loading rates for component 1 and between 95 % and 100 % of the loading rates for component 2. At the end of the test, most likely due to an incorrect sampling, the measured concentrations for both components at the concentration 18 mg/L and 32 mg/L were not plausible. However, the treatments were prepared correctly. This was confirmed by the measured start concentrations of both components and the plausible measured TOC concentration at the beginning and at the end of the test as well as the plausible inhibition values in the algae test.

In the other treatments the measured concentrations at the end of the test lay between 95 % and 119 % of the loading rates for component 1 and between 72 % and 79 % of the loading rates for component 2.

 

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 measurement. The test item concentration based on TOC measurement lay between 91 % and 101 % of the loading rates at the beginning of the test, and between 74 % and 98 % at the end of the test.

The almost identical TOC concentrationsand the measured concentrations of the both markersin 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 solutions 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 all tested loading rates for both endpoints (growth rate and yield).

At the two highest loading rates 56 mg/L and 100 mg/L, algae growth was almost completely inhibited. The three lowest loading rates showed a clear dose-response relationship.  

 

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

Endpoint	NOEL	LOEL	EL10	EL50
Growth Rate	< 10 mg/L	≤ 10 mg/L	7.96 mg/L	23.96 mg/L
Yield	< 10 mg/L	≤ 10 mg/L	7.77 mg/L*	11.62 mg/L

* extrapolated by the program