1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

N/A

Analytical monitoring:

yes

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

N/A

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Pseudokichneriella subcapitata (ID: PS-IHB-140104)
- Source (laboratory, culture collection): institute of hydrobiology, Chinese Academy of Sciences
- Method of cultivation: The algae were inoculated in 250 mL glass flasks with a concentration of 10^4 cells/mL, incubated during 3 days and then inoculated to another flask. At the beginning of the test, the algae were in the exponential growth phase when inoculated into the test solutions.


ACCLIMATION
- Acclimation period: The algae were inoculated in 250 mL glass flasks with a concentration of 10^4 cells/mL, incubated during 3 days and then inoculated to another flask. At the beginning of the test, the algae were in the exponential growth phase when inoculated into the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

no details mentioned

Test temperature:

23.2 - 23.6 °C

pH:

8.02 - 9.33

Dissolved oxygen:

not mentioned

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: 100 mg/L
measured concentration below the limit of detection (LOD = 0.214 mg/L)

Details on test conditions:

lnoculation and incubation
An inoculum culture in the test medium was pre-cultured for 3 days before start of the test under the test conditions. At the begining of the test, the alga was in the exponential growth phase when inoculated to the test solutions and the density of the alga was 9.103 x 10^5/mL. For the treatment group, 3.30 mL of the alga culture were inoculated into 300mL of test solution and the initial cell density of the test solution was about 10^4 /mL (for the control cultures, 6.59 mL of the alga culture were inoculated into 600mL of alga medium). 100mL of test solution was added into each of 250 mL glass conical flasks.

Conditions of incubation
Each flask was then capped with Parafilm and put in a culturing chamber by shaking at a speed of 130rpm.
Temperature: 23.2°C-23.6°C.
Illumination: the surface where the cultures are incubated received continuous, uniform fluorescent illumination at equivalent range of 5614-5842 lux for cool white light. Any differences from the selected light intensity over the test area did not exceed the range of ±15%.

Observation and measurements
The algal biomass in each vessel was determined by a cell counter at the beginning and the end of the test. The light density was determined at 0, 24, 48 and 72h during the test period. At the beginning of the test, pH values for the control and each test concentration were measured using the remaining test solutions. At the end of the test, one vessel was randomly taken from each treatment group and the control group for pH measurement and microscopic observation of the algae. The concentrations of the test substance were determined at 0 and 72h of the test. 10 mL of test solution was taken from one test vessel of each concentration. Before sampling, separation of algae from the test medium was made by syringe filters.

- Test vessel: sterile-aerated Erlenmeyes flasks
- Initial cells density: 10000 cells/mL
- Control end cells density: 1162833 cells/mL
- 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
Analytical grade salts were dissolved in deionised water to obtain separate alga medium stock solutions. The stock solutions were filtrated with 0.22 µm filter membrane and kept at 2-8 °C. The alga medium was prepared by diluting the stock solutions in deionised water shortly before the test began.


TEST MEDIUM / WATER PARAMETERS
no details mentioned


OTHER TEST CONDITIONS
Illumination: the surface where the cultures are incubated received continuous, uniform fluorescent illumination at equivalent range of 5614-5842 lux for cool white light. Any differences from the selected light intensity over the test area did not exceed the range of ±15%.
At the beginning of the test, pH values for the control and each test concentration were measured using the remaining test solutions. At the end of the test, one vessel was randomly taken from each treatment group and the control group for pH measurement and microscopic observation of the algae.
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Light intensity and quality: approx. 5614-5842 lux, white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell numbers for exposure times of 0, 24, 48, 72h
- Chlorophyll measurement: no
- Other: none


- Range finding study: The test substance is insoluble. A nominal concentration of 100mg/L was prepared and stirred (via magnetic stirrer) for 72h at 500rpm in the dark, and then filtered with 0.45µm nitrocellulose membrane to prepare the saturated solution under test conditions. One nominal concentration of the saturated solution was used in the range-finding test. Three replicates were included for each treatment as well as six for the control with alga medium. The test duration was 72h.

In the range-finding test, there was no statistically significant difference in alga growth between the treatment group and the control one (P=0.8199 for average specific growth rate and P=0.7855 for yield, 19.2). Therefore, a limit test would be performed.

Reference substance (positive control):

no

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

Remarks on result:

other: not toxic in the range of water solubility

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no observations made
- Any stimulation of growth found in any treatment: 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

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statistical evaluation performed

Table 1: Cell density (cell /ml) of Pseudokirchneriella subcapitata exposed to the test substance for 72h in the limit test:

Group		0h	24h	48h	72h
Blank control	1	10000	60300	379500	1435000
	2	10000	58470	366800	1623000
	3	10000	62720	370300	1554000
	4	10000	57910	391800	1686000
	5	10000	62430	357600	1538000
	6	10000	62550	346600	1609000
	Mean	10000	60730	368767	1574167
	CV (%)	0	3.56	4.3	5.48
Treatment group /saturated solution)	1	10000	63180	372300	1551000
	2	10000	60370	369100	1546000
	3	10000	59970	351500	1577000
	4	10000	62640	364400	1589000
	5	10000	58300	359500	1518000
	6	10000	59130	353100	1515000
	Mean	10000	60598	361650	1549333
	CV (%)	0	3.19	2.34	1.94

Table 2: Growth of Pseudokirchneriella subcaptitata for the blank control cultures in the limit test

Blank Control	Average specific growth rate					Yield
	0 - 24h	24 - 48h	48 - 72h	CV (%) for Section-by-section	0 - 72 h
1	1.80	1.84	1.33	17.1	1.66	1425000
2	1.77	1.84	1.49	10.9	1.70	1613000
3	1.84	1.84	1.43	12.9	1.68	1544000
4	1.76	1.91	1.46	13.5	1.71	1676000
5	1.83	1.75	1.46	11.6	1.68	1528000
6	1.83	1.71	1.54	8.9	1.69	1599000
Mean	1.80	1.80	1.54	12.5	1.69	1564167
CV (%) for replicates	1.99	4.05	4.72	-	1.10	5.51

Table 3: Growth of Pseudokirchneriella subcapitata exposed to the test substance in the limit test

Treatment group		Avergae specific growth rate					Yield
		0 -24 h	24 -48h	48 -72h	0 -72h	72 h Inhibition	0 -72h	72 h Inhibition
saturated solution	1	1.84	1.77	1.43	1.68	0.27	1541000	1.48
	2	1.80	1.81	1.43	1.68	0.33	1536000	1.80
	3	1.79	1.77	1.50	1.69	-0.06	1567000	-0.18
	4	1.83	1.76	1.47	1.69	-0.21	1579000	-0.95
	5	1.76	1.82	1.44	1.67	0.69	1508000	3.59
	6	1.78	1.79	1.46	1.67	0.74	1505000	3.78
	Mean	1.80	1.79	1.46	1.68	0.29	1539333	1.59
	CV (%)	1.76	1.32	1.93	0.38	-	1.95	-

                                                                           

Validity criteria fulfilled:

yes

Remarks:

mean biomass control cultures increased 157 fold, mean coefficient of variation section-by-section specific growth rates control cultures 12.5%, coefficient of variation of average specific growth rates test period in replicate control cultures 1.1 %

Conclusions:

Not toxic to algae under test conditions in the range of water solubility.

Executive summary:

The growth inhibition effect of 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters to Pseudokirchneriella subcapitata was determined in a 72h test according to OECD 201.

A nominal concentration of 100 mg/L of the test substance was prepared and stirred for 72 h at 500 rpm in the dark followed by filtration using a 0.45 µm nitrocellulose membrane to make a saturated solution of test system (referred to as "saturated "solution" in this report, since the measured concentration was below LOD, LOD = 0.214 mg/L). Based on the results of the range-finding test, one treatment group of saturated solution and one control group of alga medium were included in the limit test. Six replicates for each group were run. The duration was 72h.

The mean biomasss of the control cultures was increased by 157 folds, which is higher than the validity criterion of at least 16 within the 72h test period., corresponding to a specific growth rate of 0.92 day^-1 . The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 12.5 %, which was below 35 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.1%, which was below 7 %. Hence, the test was cosidered to be valid. During the test, the measured concentrations of the tst substance analyzed by gas chromatography were below the limit of detection (LOD, LOD=0.214 mg/L).

Under the present test conditions, no inhibition effects were observed on Pseudokirchneriella subcapitata exposed to the test substance. Therefore, based on average specific growth rate, the 72h NOEC was not less than the measure concentration of the saturated solution, the LOEC were also higher than the measured concentration of the saturated solution. The concentration was below LOD, LOD=0.214 mg/L.

Not toxic to algae under test conditions in the range of water solubility.

Description of key information

1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters under test conditions has shown no toxic effects to Pseudokirchneriella subcapitata in the range of water solubility.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The growth inhibition effect of 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters to Pseudokirchneriella subcapitata was determined in a 72h test according to OECD 201.

A nominal concentration of 100 mg/L of the test substance was prepared and stirred for 72 h at 500 rpm in the dark followed by filtration using a 0.45 µm nitrocellulose membrane to make a saturated solution of test system (referred to as "saturated "solution" in this report, since the measured concentration was below LOD, LOD = 0.214 mg/L). Based on the results of the range-finding test, one treatment group of saturated solution and one control group of alga medium were included in the limit test. Six replicates for each group were run. The duration was 72h.

 

The mean biomasss of the control cultures was increased by 157 folds, which is higher than the validity criterion of at least 16 within the 72h test period., corresponding to a specific growth rate of 0.92 day-1 . The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 12.5 %, which was below 35 %. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.1%, which was below 7 %. Hence, the test was cosidered to be valid. During the test, the measured concentrations of the tst substance analyzed by gas chromatography were below the limit of detection (LOD, LOD=0.214 mg/L).

 

Under the present test conditions, no inhibition effects were observed on Pseudokirchneriella subcapitata exposed to the test substance. Therefore, based on average specific growth rate, the 72h NOEC was not less than the measure concentration of the saturated solution, the LOEC were also higher than the measured concentration of the saturated solution. The concentration was below LOD, LOD=0.214 mg/L.