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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 2017 to April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations an frequency: Samples were taken from all test concentrations and the control at t=0h, t=24 h and t=48h.
- Sampling method: 2.0 mL from the approximate center of the test vessels. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the highest item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION:
The batch of 1,3-Bis (4-hydroxy benzoyl) benzene tested was an off white crystalline powder with a purity of 99.53% which was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying 15 minutes of ultrasonic waves followed by 80 minutes of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10x4 cells/mL.
Any residual volumes were discarded
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata),
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use

ALGAE CULTURE
- Stock culture : Algae stock cultures were started by inoculating growth medium 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
- Culturing media and conditions: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Light Intensity : 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
Between 22 and 23°C
pH:
From 8.0 to 8.4
Nominal and measured concentrations:
Solutions containing 1.0, 10, and 100% of a Saturated Solution (SS) prepared at a loading rate of 100 mg/L.
(Range Finding test: Solutions containing 1.0, 10 % of a SS prepared at a loading rate of 100 mg/L and Limit test: 100 % of a SS prepared at a loading rate of 100 mg/L).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Renewal rate of test solution: none static
- Initial cells density: An initial cell density of 1 x 104 cells/mL
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- No. of vessels per concentration (replicates): 6 replicates of the control and the highest test concentration, 3 replicates of each intermediate test concentration, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

TEST MEDIUM / WATER PARAMETERS
- Medium : M2
- pH : At the beginning and at the end of the test in the control and the highest test concentration.
- Temperature of medium : Continuously in a temperature control vessel.
- Appearance of the cells : At the end of the test, microscopic observations were performed on the control and the limit concentration to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod, Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 85 to 87 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period

TEST CONCENTRATIONS:
- Range finding test: Solutions containing 1.0, 10 % of a SS prepared at a loading rate of 100 mg/L.
- Limit test: 100 % of a SS prepared at a loading rate of 100 mg/L).
- Results used to determine the conditions for the definitive study: not needed
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Measured Concentrations:
Samples taken from the highest test concentration were analysed i.e. undiluted SS (Saturated Solution). At the start of the test, the actual exposure concentration was 0.36 mg/L. This concentration decreased to 0.24 mg/L at the end of the test. Based on this result, the TWA concentration was 0.28 mg/L in the undiluted SS (Saturated Solution).

- Inhibition of Growth Rate and Inhibition of Yield:
No inhibition of algal growth was observed at the end of the test.

The group mean growth rates and the percentages of growth rate inhibition (total test period) are summarized Table 1
The group mean yields and the percentages of yield inhibition are summarized in Table 2.

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest concentration when compared to the control.
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

No EC10 and EC50-values could be calculated because the test item proved to be non-toxic (EC10 and EC50 > maximum solubility of the test item in test medium).

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

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

1,3-Bis (4-hydroxy benzoyl) benzene.

%SS prep. at a loading rate of 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

1.954

0.0251

6

 

1.0

1.924

0.0088

3

1.5

10

1.941

0.0241

3

0.7

100

1.946

0.0184

6

0.4

 

Table 2 : Yield And Percentage Inhibition For The Total Test Period

1,3-Bis (4-hydroxy benzoyl) benzene.

%SS prep. at a loading rate of 100 mg/L

Mean

Std. Dev.

n

%Inhibition

Control

351.1

26.14

6

 

1.0

320.2

8.39

3

8.8

10

337.4

24.91

3

3.9

100

342.5

18.23

6

2.4

 

Validity criteria:

1.       In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 352).

2.       The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 20%).

3.       The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.3%).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene tested.
The EC50 for both growth rate and yield inhibition (72h-ErC50 and 72h-EyC50, respectively) was beyond the range tested, i.e. exceeded a TWA concentration of 0.28 mg/L being considered to be the maximum soluble concentration of the test item in test medium.
The 72h-NOEC for both growth rate and yield inhibition was 0.28 mg/L, based on both statistical significance and biological relevance.

Executive summary:

The objective of the study was to evaluate 1,3-Bis (4-hydroxy benzoyl) benzene for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50 for both inhibition of growth rate and inhibition of yield.

The study was compliant with the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, as the 1,3-Bis (4-hydroxy benzoyl) benzene was poorly soluble in test medium, procedures were also based on the test methods described in the OECD series on testing and assessment number 23, 2000.

 

Thus a Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and the undiluted SS in the limit test. In addition, three replicates per group were exposed to test solutions containing 1.0 and 10% of the SS prepared at a loading rate of 100 mg of1,3-Bis (4-hydroxy benzoyl) benzeneper litre in a combined range-finding test. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.

Samples taken from the undiluted SS were analysed. At the start of the test, the actual exposure concentration was 0.36 mg/L. This concentration decreased to 0.24 mg/L at the end of the test. Based on this result, the TWA concentration was 0.28 mg/L in the undiluted SS.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene tested.

The EC50 for both growth rate and yield inhibition (72h-ErC50 and 72h-EyC50, respectively) was beyond the range tested, i.e. exceeded a TWA concentration of 0.28 mg/L being considered to be the maximum soluble concentration of the test item in test medium.

The 72h-NOEC for both growth rate and yield inhibition was 0.28 mg/L, based on both statistical significance and biological relevance.

Description of key information

The toxicity of the 1,3 -Bis (4 -hydroxy benzoyl) benzene to the freshwater green algae Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 201. In addition, due to the low solubility of the test item, procedures were based on the test methods described in the OECD series on testing and assessment number 23 (2000).

Under the conditions of the study, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene tested. The EC50for both growth rate and yield inhibition (72h-ErC50and 72h-EyC50, respectively) was beyond the range tested, i.e. exceeded a TWA concentration of 0.28 mg/L being considered to be the maximum soluble concentration of the test item in test medium at a loading rate of 100 mg/L. The 72h-NOEC for both growth rate and yield inhibition was 0.28 mg/L.

Key value for chemical safety assessment

Additional information

The toxicity of the test item to freshwater green algae Pseudokirchneriella subcapitata was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.