D-cellobiose

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 13, 2022 - ongoing

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Rapid degradation in this study, which may be specific to the medium used, reduces the reliability of this study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 23, 2006

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

LOT No.: L1021174
Appearance: white, dry powder
Expiry date: 28 October 2023
Storage condition: at room temperature, protected from light
Safety precautions: according to MSDS
Purity: 99.54 %

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1-100 µg/mL

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion without using any solubilising agent, as the substance is well water soluble.
A stock solution of 100 mg/L nominal concentration was first prepared by dissolving an amount of 0.1217 g test item in 1217 mL dilution water (OECD Medium). Test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solution. After the formulation procedure the algal cells were immediately introduced into the test solutions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: Raphidocelis subcapitata (also known as Pseudokirchneriella subcapitata)
- Source (laboratory, culture collection): Experimental Phycology and Culture Collection of Algae, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.

ACCLIMATION
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2 - 4 days. The pre-culture was incubated for three days at this test. The algal cultures used in this study did not contain deformed or abnormal cells.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted algal growth medium (OECD Medium, according to OECD 201

Total exposure duration:

72 h

Test temperature:

The temperature was between 23.0 and 23.4 °C measured in the flask and in the range of 22.4 – 23.7 °C measured within the climate chamber.

pH:

7.56 – 8.53

Nominal and measured concentrations:

Based on the results of the preliminary range-finding test the following five nominal test concentrations in a geometric series with a spacing factor of 2 was used in the main test:
6.25, 12.5, 25, 50 and 100 mg/L. Untreated control group ran in parallel.

The corresponding calculated exposure concentrations (based on analytical measurements; see details in section 8.2) were the followings:
0.99, 1.80, 2.74, 5.04 and 9.97 mg/L.
Biological results and endpoints are based on the measured concentrations (i.e. calculated exposure concentrations).

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Erlenmeyer flasks of 250 mL volume
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density: Cell density increased by a factor of 95.5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: -

TEST MEDIUM / WATER PARAMETERS
- test medium: Reconstituted algal growth medium (OECD Medium, according to OECD 201)
- Source/preparation of dilution water: deionised water (prepared in TOXI-COOP ZRT. by BWT Christ Ministil and RiOs 5 Smart water purification systems)


OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Adjustment of pH: No
- Photoperiod: 12/12
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks at the start of the test was 7391 lux, which was ensured with fluorescent lamps (wavelength range: 400 – 700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: manual cell counting using microscope in each testing flask during the 72-hour test, at 24-hour intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes, with test concentrations go 0.01, 0,1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 21.86 mg/L

95% CI:

>= 14.96 - <= 47.25

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC50 is extrapolated, but is clearly higher than the highest measured test concentration (9.97 mg/L)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 3.09 mg/L

95% CI:

>= 1.88 - <= 4.05

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.6 mg/L

95% CI:

>= 3.83 - <= 5.68

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.56 mg/L

95% CI:

>= 0.92 - <= 2.08

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L plus a concurrent control were tested and analysed from samples taken at the start and 24-h intervals thereafter during the test. The measured concentrations deviated more than 20 % from the nominal during the experiment (as the test item was not stable in OECD medium), therefore, exposure concentrations were calculated based on the analytical measurements in order to express the biological endpoints relative to the measured concentrations. As no reliable analytical data (too high variability between the test groups) was obtained on the third analytical occasion (at 48 h), only the data obtained from the samples taken at the start and at the end of the test was used for calculation in order to determine more exact exposure concentrations. However, by the end of the test period (72 h), measured concentrations were below the analytically measurable range (i.e. < LOQ) in each test group. For that case, a value of half of the LOQ (LOQ/2 = 0.005 mg/L) was used for calculation according to the recommendation of OECD 23 Guidance document (ENV/JM/MONO(2000)6/REV1), section 7.11.2.3 [6]. This high concentration decline was considered to be due to the characterisation of the test item and its behaviour in OECD medium, which was confirmed by the results of the analytical method validation study (Study number: 123-100-6540).

 

Validity criteria fulfilled:

yes

Conclusions:

In a GLP-compliant study according to OECD TG 201 with Raphidocelis subcapitata, cellobiose reduced algae growth with a measured EC50 of 21.86 mg/L.

Executive summary:

Exponentially-growing cultures of Raphidocelis subcapitata were exposed to cellobiose under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the control, respectively. The algae cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. Glass 250 mL Erlenmeyer flasks filled up with 100 mL test liquid were used as test vessels. The algae cell concentration was determined by manual cell counting using microscope in each testing flask during the 72-hour test, at 24-hour intervals.

For determination of the test item concentrations, samples were taken from each test concentration and from the control at each day of the test. Concentrations were determined by HPLC-MS/MS method.

72-h EC₁₀, EC₂₀, EC₅₀, NOEC and LOEC were determined for both response variables (average specific growth rate and yield).

Biological results and endpoints are based on the measured concentrations (i.e. calculated exposure concentrations). All validity criteria were met and therefore, the study was considered to be valid.

The following results were obtained:

Endpoints (0-72 h)	Growth rate [mg/L]	Yield [mg/L]
	Calculation based on measured concentrations
EC10	3.09	1.56
95 % conf. limits	1.88 – 4.05	0.92 – 2.08
EC20	6.05	2.26
95 % conf. limits	4.78 – 7.54	1.57 – 2.82
EC50	> 9.97* (21.86 calculated)	4.60
95 % conf. limits	14.96 – 47.25	3.83 – 5.68
LOEC	2.74	2.74
NOEC	1.80	1.80

 

Description of key information

In a GLP-compliant study according to OECD TG 201 with Raphidocelis subcapitata, cellobiose reduced algae growth with a measured EC50 of 21.86 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

21.86 mg/L

EC10 or NOEC for freshwater algae:

1.8 mg/L

Additional information

Rapid degradation in this study, which may be specific to the medium used, reduces the reliability of this study.