Plum, ext.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 August 2018 - 28 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Plum, ext.

EC Number:

290-179-3

EC Name:

Plum, ext.

Cas Number:

90082-87-4

Molecular formula:

not applicable

IUPAC Name:

Extract obtained from fruits of Prunus domestica L. Rosaceae

Test material form:

liquid

Details on test material:

- Physical appearance: Blackish brown to dark brown liquid
- Storage conditions: At room temperature

Specific details on test material used for the study:

- For the combined limit/range-finding study a different batch of the test item was used, this did not affect the outcome of the final study.
- No correction factor for purity is required and therefore not applied
- The test item is soluble and stable in water

Sampling and analysis

Analytical monitoring:

yes

Remarks:

TOC analysis

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control from vessels without algae:
- Frequency: at t=0 h and t=72 h
- Sampling volume: 40 mL
- Sample storage conditions before analysis: Samples were stored in a refrigerator (2-8°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying a 15- minute period of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Evidence of undissolved test material: the highest test concentration was clear and of very slight yellow coloration, while the lower test concentrations were clear and colourless.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 d (pre-culture)
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C, until the pre-culture started.

PRE-CULTURE:
- Pre-culture method: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2; according to the OECD 201 Guideline, formulated using Milli-RO water) at a cell density of 1 x 10^4 cells/mL.
- Culturing media and conditions (same as test or not): Yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO2/L

Test temperature:

22-24°C

pH:

At the start of the test: 8.0 - 8.2
At the end of the test: 8.2-8.4

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
Measured TOC concentrations: A clear increase of TOC concentrations was found at the start of the test with increasing nominal test item concentration ranging from 0.23 to 16 mg TOC/L, indicating a correct preparation of the test solutions (see table 1).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped vessels
- Test solution volume: 50 mL
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 71 x10^4
- No. of replicates:
3 replicates of each test concentration;
6 replicates of the control;
2 replicates of each test concentration without algae for sampling purposes at the end of the test.

GROWTH MEDIUM
- Standard medium used: yes (M2, according to OECD 201 Guideline)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 test medium, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Stock culture medium: M1, pre-culture medium: M2.
- Intervals of water quality measurement: pH at the beginning and at the end of the test; Temperature of medium continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: TLD-lamps with a light intensity within the range of 84 to 86 µE.m^2.s^1.
- Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Throughout the test, cells were counted using a microscope and a counting chamber. In the combined limit/range-finding study cell densities were also determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). However, because of precipitation the spectrophotometric method very likely overestimated the number of cells and this method was therefore not used for the final test.
- Appearance of the cells: At the end of the final test microscopic observations were performed on all test concentrations and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Six replicates of exponentially growing algae were exposed to a control and a nominal test concentration of 100 mg/L and three replicates of exponentially growing algae were exposed to test concentrations of 0.10, 1.0 and 10 mg/L. At a concentration of 100 mg/L growth rate was inhibited by 5.8%. Baed on these results the test was repeated as a limit test at a concentration of 100 mg/L. The limit test showed 21% inhibition of growth but growth inhibition was possibly overestimated because of algae clumps. Therefore, a full final test was performed.

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (Potassium dichromate; July 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on statistical significance

Details on results:

Results of TOC analysis: the TOC of the test item was 31.78%, this value was used to estimate nominal TOC concentrations in the final test. A clear increase of TOC concentrations was found at the start of the test with increasing nominal test item concentration ranging from 0.23 to 16 mg TOC/L, indicating a correct preparation of the test solutions. Since TOC-analysis is a non-specific method, the effect parameters were determined based on nominal concentrations.

Results of final study: Growth rate was not statistically significant inhibited at nominal concentrations of 1.0, 3.2, 10 and 32 mg/L, while a statistically significant inhibition of 6.6% was observed at 100 mg/L. Because no biologically relevant inhibition of growth (10%) was observed, the NOEC for biologically relevant inhibition was determined to be 100 mg/L. ErC50 and ErC10 values were determined to be higher than the highest tested concentration (> 100 mg/L).

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to nominally 1.0 and 3.2 mg/L when compared to the control. Algal cells exposed to nominally 10 mg/L and higher were found to form clumps and were smaller and misshaped compared to algal cells in the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Test concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50: The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L.
- The results were within the historical range of the test facility.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

No ECx-values could not be determined because the observed effects were below 10%.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 2 Growth Rate and Percentage Inhibition for the Total Test Period

Plum concentrate Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.416	0.0499	6
1.0	1.423	0.0074	3	-0.50
3.2	1.465	0.0582	3	-3.5
10	1.452	0.0652	3	-2.6
32	1.396	0.0315	3	1.4
100	1.323	0.0211	3	6.6 *

* Effect was statistically significant but biologically not relevant (<10%).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'

Conclusions:

The 72h-ErC50 and 72h-ErC10 of Plum concentrate towards aquatic algae was determined to exceed the highest nominal tested concentration (> 100 mg/L). The 72h-NOEC for growth rate inhibition was 32 mg/L based on statistical significance and 100 mg/L based on biological relevance.