Registration Dossier

Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dibutyl terephthalate (DBT)
- Substance type: production sample
- Physical state: viscous liquid
- Analytical purity: 99.32%
- Lot/batch No.: TX061106
- Storage condition of test material: stored at room temperature in the original container in a dark ventilated cabinet

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At test initiation and 24 hours, the entire contents (100 mL) were removed from two test vessels and one control and solvent control vessel and analyzed for DBT concentration. At test termination (72 hours), the contents of two sets of three replicate flasks were composited and sampled in entirety for the treatment and the contents of one set of three flasks was composited and sampled in entirety for the control and solvent control.Additionally, at 24 and 72 hours of exposure, the entire contents of a flask which contained no algae was removed at each interval from the 0.010 mg a.i./L test concentration. The results of the analyses were compared with those obtained for the 24- and 72-hour analyses of the 0.010 mg a.i./L solutions containing algae to assess the impact that algae had on the test substance concentration.

Algal growth was monitored at each subsequent 24-hour interval. A single cell count was conducted on each replicate solution of the treatment and the control using a hemacytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. One or more hemacytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of solution, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval.

Test solutions

Vehicle:
yes
Details on test solutions:
A 0.10 mg a.i./mL stock solution was prepared five days prior to test initiation by placing 0.0101 g of DBT (0.0100 g as active ingredient) in a 100-mL volumetric flask and bringing it to volume with acetone. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. The stock solution was refrigerated at 2 to 8 °C when not in use. Since the test substance is known to adsorb to glass, the test vessels were pre-acclimated to 0.010 mg a.i./L test solutions for two days prior to test initiation. The 0.010 mg a.i./L preacclimation solutions were prepared directly in 15 test vessels by diluting 0.015 mL of the 0.10 mg a.i./mL primary stock solution to a final volume of 150 mL with AAP medium. The first
pre-acclimation solutions were prepared in the morning two days prior to test initiation, and later that day, emptied and prepared again. The same procedure was followed for the following day of pre-acclimation. The vessels were maintained under test conditions (e.g., temperature, lighting and shaking) in the environmental chamber during the pre-acclimation phase. At test initiation, the pre-acclimation solutions were emptied from the test flasks and the test solutions were prepared in each of 15 flasks by adding 0.010 mL of the 0.10 mg a.i./mL stock solution to 100 mL of AAP medium per test flask to yield a final test solution concentration of 0.010 mg a.i./L. The flasks were then placed on the shaker table and shaken for approximately
1.5 hours to promote mixing. After mixing the test solutions were observed to be clear and colorless and did not contain any visible undissolved material. Solvent control flasks were prepared by added 0.010 mL of acetone to 100 mL of AAP in each of 13 flasks. Thirteen control flasks were also prepared and contained only 100 mL of AAP medium. Each test flask was labeled with the test concentration, replicate, test species and study number. All test vessels were fitted with stainless steel caps which permitted gas exchange.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The alga used in this toxicity test was the freshwater green alga, Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum, strain 1648, Class Chlorophyceae. The alga was obtained from University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers. The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 C and continuous illumination at the surface of the medium with an intensity range of 4440 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test with DBT was taken from a stock culture that had been transferred to fresh medium four days before testing.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
Continuous temperature monitoring established that the temperature ranged from 23 to 24 ºC during the test period.
pH:
The pH of the test solutions ranged from 6.8 to 6.9 at test initiation. At test termination, the test solution pH ranged from 7.3 to 8.9. The increase in pH during the exposure is common in static algal cultures and is due to photosynthesis by the algae.
Salinity:
Conductivity measured at test initiation and termination in the treatment and control solutions ranged from 80 to 90 μmhos/cm.
Nominal and measured concentrations:
The results of the analysis of the exposure solution for DBT concentration are presented in Table 1. At 0 hour, replicate measured concentrations of DBT in the test solutions, 0.011 and 0.014 mg a.i./L, closely approximated the desired nominal concentration. The concentrations of DBT decreased at 24 and 72 hours of exposure. The 72-hour test sample results were below detectable limits (< 0.00013 mg a.i./L). Since the concentration of DBT decreased rapidly during the test, the exposure concentration is expressed as the average initial measured concentration measured on day 0. The OECD Guideline #201 allows the use of the initial measured concentration under these circumstances. The analytical result of the 24-hour sample from the 0.010 mg a.i./L nominal treatment level, with algae present, was 0.0054 mg a.i./L. At 24 hours, the equivalent test solution without algae present resulted in a recovery of 0.0084 mg a.i./L, indicating that the presence of algae may have had a slight impact on the concentration of DBT in the test solution. The analytical results of the 72-hour sample from the 0.010 mg a.i./L solution without algae was below the detection limit for this sample (< 0.00038 mg a.i./L). The 72-hour results from the 0.010 mg a.i./L solution with algae present was also below the limit of detection (< 0.00013 mg a.i./L). Therefore, the decline in test substance concentration appears not to be related to the presence of algae.
Details on test conditions:
The test was conducted in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 23 ± 2 oC, continuous light intensity of 4440 to 5900 lux (420 to 550 footcandles) and photosynthetically-active radiation (PAR) range of 60 to 120 μE/m2/s. Two orbital shaker tables were required and provided a shaking rate of 100 ± 10 rpm. Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured at four locations around the perimeter of the shaker tables with a VWR Traceable light meter at 0 hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a
Licor Model LI-189 photometer and probe (Model LI-190SA). Test flasks were randomly placed on two shaker tables at test initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the test solution present in individual treatment, control and solvent control flasks prepared for this purpose. At test termination, after cell counts were completed, the replicate solutions for each treatment and the control were respectively composited for pH and conductivity measurements. Test solution pH was measured with a Jenco Model 60 pH meter, and conductivity was measured with a Yellow Springs Instrument (YSI) Model No. 33 salinityconductivity- temperature meter.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.013 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Inhibition of 14 and 3% was observed for 72-hour total yield and growth rate, respectively, relative to the solvent control data. Therefore, the EC50 values for 72-hour yield and growth rate are greater than the average initial measured concentration of 0.013 mg a.i./L. Since the average initial measured concentration, 0.013 mg a.i./L, closely approximated the functional limit of solubility of dibutyl terephthalate (DBT) in algal medium, additional testing to further define the EC50 values was not performed. The OECD Guideline #201 (OECD, 2006) suggests the EC20 value may be used as the No-Observed Effect Concentration (NOEC). Growth inhibition in this test did not exceed 14%.

Any other information on results incl. tables

Table 1:  Concentrations of DBT (mg a.i./L)      
Nominal  Measured @ 0 Hours   Measured @ 24 Hours    Measured @ 72 Hours  
Control  <0.00039  <0.00038   <0.00013
 Solvent Control  <0.00039  <0.00038  <0.00013
0.010   0.011  0.0043  <0.00013
0.010 0.014  0.0065  <0.00013 
 Mean 0.013   0.0054 <0.00013
QC#1 0.000500  0.000539 (108%)a  0.000542 (108%)a  0.000595 (119%)a 
QC#2 0.00500  0.00590 (118)a  0.00552 (110)a   0.00591(118)a
QC#3 0.0600   0.0660 (110)a  0.0553 (92.2)a  0.0675 (112)a
 a = The percent recovery for the corresponding QC sample is presented in parentheses.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Well conducted GLP study conducted with a difficult substance.
Executive summary:

A 72-Hour Acute Toxicity Test with Freshwater Green Alga, Pseudokirchneriella subcapitata, was conducted on dibutyl terephthalate. The test was conducted as a limit test at a concentration slightly above the measured water solubility of the substance. Inhibition of 14 and 3% was observed for 72-hour total yield and growth rate, respectively, relative to the solvent control data. Therefore, the EC50 values for 72-hour yield and growth rate are greater than the average initial measured concentration of 0.013 mg a.i./L. Since the average initial measured concentration, 0.013 mg a.i./L, closely approximated the functional limit of solubility of dibutyl terephthalate (DBT) in algal medium, additional testing to further define the EC50 values was not performed. The OECD Guideline #201 (OECD, 2006) suggests the EC20 value may be used as the No-Observed Effect Concentration (NOEC). Growth inhibition in this test did not exceed 14%.