Triphenyl phosphate

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish juvenile: (sub)lethal effects

Remarks:

Fish Sexual Development Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

biological test: 19.06.-02.09.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 234 (Fish Sexual Development Test)

Version / remarks:

28.07.2011

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Triphenyl phosphate

EC Number:

204-112-2

EC Name:

Triphenyl phosphate

Cas Number:

115-86-6

Molecular formula:

C18H15O4P

IUPAC Name:

triphenyl phosphate

Test material form:

solid: pellets

Sampling and analysis

Analytical monitoring:

yes

Remarks:

method was validated according SANCO/3029/99 rev. 4.

Details on sampling:

- Concentrations:
- Sampling method: direct sampling in aquaria (two aliquotes a 5 mL), weekly
- Sample storage conditions before analysis: part of samples were directly measured, remaining amounts were stored at <= - 18 °C as retain samples until test end.

Test solutions

Vehicle:

no

Remarks:

Initial stock solution of test item was prepared in aceton. Vehicle was completely evaporated from aqueous stock solution.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: dilution in test medium, multiple steps necessary to prepare stock solutions for flow through application, test contrations were adjusted by flow rate of stock solution and dilution water
- Eluate: -
- Differential loading: -
- Controls: -
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): used acetone was completely evaporated from aqueous stock solution
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): -
- Test concentration separation factor: √10
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
- Other relevant information:

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain origin: West Aquarium GmbH, 37431 Bad Lauterberg, Germany
- Source: Test facility bred, IME, Auf dem Aberg 1, Schmallenberg
- Life stage: fertilized eggs, obtained from individuals reared in the laboratory
- Age at study initiation (mean and range, SD): -
- Length at study initiation (length definition, mean, range and SD): -
- Weight at study initiation (mean and range, SD): -
- Method of breeding:
Parental fish for egg production were hold in aquaria with a total volume of 150 L. Holding water was of the same quality as used in the test (purified drinking water, see below). The maximum age for parental fish was 2 years. Stock density was approximately 1 g/L volume. Holding temperature was 26 °C ± 2 °C. Light/dark cycle was 12 h/12 h. Flow through rate was adjusted to reach approx. one total exchange of water per day. Fish were fed daily ad libitum with TetraMin® Hauptfutter (Tetra Werke, Melle, Germany) and brine shrimp nauplii (Artemia salina).
The broodstock was visually checked every day for mortality, illness, parasites or abnormal behavior. No prophylactic treatment of fish took place. Only healthy fish without diseases and abnormalities were used as parental fish for the production of fertilized eggs.

- Pre-exposure reproductive information

ACCLIMATION
- Acclimation period: not applicable
- Acclimation conditions (same as test or not):not applicable
- Type and amount of food during acclimation:not applicable
- Feeding frequency during acclimation: not applicable
- Health during acclimation (any mortality observed): not applicable

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable

FEEDING DURING TEST
From approximately day 14 pf onwards, brine shrimp nauplii (Artemia salina) were added twice daily. From day 14 pf onwards, ground TetraMin flakes were added once daily to the fish feed. From day 14 pf onwards, larvae were allowed to freely move to the main vessel in order to support undisturbed growth.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
not stated
- Method of collection of fertilised eggs:
glass spawning-tray covered with stainless steel lattice to protect eggs, an artificial substrate was attached to stimulate spawning
- Subsequent handling of eggs:
collected eggs were transferred from the spawning-tray into a sieve, rinsed with clean water in order to remove any debris and then put into glass dishes. Fertilized eggs (microscopic determination of > four cell stage, i.e. early blastula stage) were transferred by means of a widened and de-burred pipette tip into the test chambers. Time from spawning until transferring into the test solutions was kept as short as possible, and no later than 12 h after fertilization
- Subsequent handling of juvenile fish: not applicable

POST-HATCH FEEDING
- Start date: 4pf
- Type/source of feed: ground larval diet (TetraMin Baby, Tetra Werke, Melle, Germany) and liquid rearing feed (Nobil fluid, JBL, Neuhofen, Germany)
- Amount given: not stated
- Frequency of feeding: twice daily

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

73 d

Remarks on exposure duration:

The exposure was prolonged to 73 dpf in order to allow complete maturation of gonads.

Test conditions

Hardness:

total: 1.0-1.2 mmol

Test temperature:

27.1 °C - 27.6 °C

pH:

mean pH value: 7.59 - 7.80

Dissolved oxygen:

mean saturation: 86-93 %ASV

Conductivity:

256-275 µS/cm

Nominal and measured concentrations:

nominal: 1.0, 3.2, 10.0, 32.0, 100 µg/L ;
mean measured: 1.11, 3.01, 7.76, 33.3, 76.8 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria with two fry cages, being glass cylinders with a brim height of 10 cm and a diameter of 8 cm. The bottom of each cage was a Teflon gaze with a pore size of 0.4 mm.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 28 L total volume, approx. 25 L fill volume
- Aeration: not necessary, continuos water exchange
- Type of flow-through (e.g. peristaltic or proportional diluter): dosage system with membrane pumps and mixing chamber
- Renewal rate of test solution (frequency/flow rate): 5 volumes per day
- No. of organisms per vessel: 2 x 15 fertilized and randomized eggs
- No. of organisms per mL or well: -
- No. of vessels per concentration (replicates): 4 ( + 2 at highest test concentration for examination of impacts on heart tissue)
- No. of vessels per control (replicates): 4 ( + 2 for examination of impacts on heart tissue)
- Vehicle control performed: no
- No. of vessels per vehicle control (replicates): -
- Biomass loading rate: -
- Stocking density: -
- No. of independent and valid run (solutions and spawn): -

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified tap water
- Total organic carbon:0.3023-0.9582 mg/L
- Particulate matter: -
- Metals: additional information in any other information on materials and methods
- Pesticides: -
- Chlorine: 0.02-0.03 mg/L (total residual)
- Alkalinity: 1.6-1.9 mmol/L
- Ca/mg ratio: 3:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: monthly

OTHER TEST CONDITIONS
- Adjustment of pH: -
- Photoperiod: 12 hours light / 12 hours dark (light intensity of approximately 1000 lux, measured 5 cm above the water surface in the middle of the test vessel
- References and settings of the spectrofluorometer or fluorescence microscope used for quantification: not stated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Time to hatch / Hatching success (2 pf to 6 pf)
post-hatch survival/length (35 pf)
Survival / length and weight / sex ratio / gonad histopathology / Liver examination / Vitellogenin content / 11-keto testosterone and 17-beta estradiol concentration / heart tissue examination (73 pf)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Justification for using less concentrations than requested by guideline: -

RANGE FINDING STUDY
- Test concentrations: nominal: 1.0, 10 and 100 µg/L; mean measured: 0.93, 9.18 and 88.3 µg/L
- Results used to determine the conditions for the definitive study: slight decrease in survival was detected in highest test concentration. Therefore, this concentration was selected as highest concentration in the main test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

3.01 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Effect concentrations 2

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

7.76 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Effect concentrations 3

Duration:

35 d

Dose descriptor:

NOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 4

Duration:

35 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 5

Key result

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

3.01 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Effect concentrations 6

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

7.76 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Effect concentrations 7

Key result

Duration:

73 d

Dose descriptor:

LC10

Effect conc.:

4.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: post-hatch survival

Effect concentrations 8

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

7.76 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 9

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Effect concentrations 10

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

males

Effect concentrations 11

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

males

Effect concentrations 12

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

females

Effect concentrations 13

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

females

Effect concentrations 14

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

7.76 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Effect concentrations 15

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Effect concentrations 16

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

males

Effect concentrations 17

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

males

Effect concentrations 18

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

7.76 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

females

Effect concentrations 19

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

wet body weight

Remarks:

females

Effect concentrations 20

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

>= 76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Effect concentrations 21

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

>= 76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks:

males

Effect concentrations 22

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

>= 76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks:

females

Effect concentrations 23

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

>= 76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: 11-keto testosterone, males

Effect concentrations 24

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

33.3 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: 17-beta estradiol level, females

Effect concentrations 25

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: 17-beta estradiol level, females

Effect concentrations 26

Duration:

73 d

Dose descriptor:

LOEC

Effect conc.:

76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

gonadal histology

Remarks:

testis stage

Remarks on result:

other:

Remarks:

significant; Students t-test, α=0.05; two-sided

Effect concentrations 27

Duration:

73 d

Dose descriptor:

NOEC

Basis for effect:

gonadal histology

Remarks:

ovary stage

Remarks on result:

not determinable

Remarks:

not significant; Students t-test, α=0.05; two-sided

Effect concentrations 28

Duration:

73 d

Dose descriptor:

NOEC

Basis for effect:

gonadal histology

Remarks on result:

not determinable

Remarks:

not significant, Students t-test, α=0.05; two-sided

Effect concentrations 29

Duration:

73 d

Dose descriptor:

NOEC

Effect conc.:

>= 76.8 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

histopathology

Remarks:

liver examination

Details on results:

For the determination of the effect contrations for the stages of the ovaris and testis as well as the histopathological findings in the gonads only the concentrations of 3.01 and 76.8 were included in analysis. Thus, no concentration-response relationship could be tested, allowing only individual statistics separately for each test level compared to controls.
For further details please see attached documents.

Reported statistics and error estimates:

For each endpoint, the NOEC and LOEC were determined. All statistics were calculated using ToxRat® Professional 3.3.

For NOEC / LOEC-determination, quantal data were arcsine-transformed prior to analysis. No Observed Effect Concentrations (NOEC) were calculated, using ANOVA, followed by Williams test or respective non-parametric approaches (e.g. Jonckheere-Terpsta test).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see attached document "validity criteria"

Conclusions:

Based on the endpoint post-hatch survival at test termination, the following NOEC was determined:

NOEC = 3.01 μg triphenyl phosphate/L (mean measured concentration),

corresponding to

NOEC = 3.20 μg triphenyl phosphate/L (nominal concentration).

The LC10 was determined to be 4.80 µg triphenyl phosphate/L (mean measured; CL= 2.77 µg/L to 7.13 µg/L).

No endocrine-related effect of the test item triphenyl-phosphate was identified.

Executive summary:

A fish sexual development test (FSDT) with zebrafish (Danio rerio) was performed to assess the potential endocrine activity and adverse effects of continuous exposure to the test item on the early life stages and sexual differentiation. An effect of the test item triphenyl phosphate on post-hatch survival at test end was observed. The NOEC for this endpoint was determined to be at 3.01 µg triphenyl phosphate/L (mean measured concentration; Williams t-test, one-sided smaller, p<0.05). Additionally, the LC10 was determined to be 4.80 µg triphenyl phosphate/L (mean measured), with a confidence interval between 2.77 µg/L and 7.13 µg/L. Mortality was mainly observed during the early life stage of zebrafish until 35 dpf, with only few cases of mortality between 35 dpf and test termination.
During the early life stage, reduced growth in terms of total length was observed at the highest test concentration of 76.8 µg triphenyl phosphate/L (mean measured). This reduced size was likely due to the general systemic toxicological effect of the test item on zebrafish during the early life stage.
However, due to the reduced number of fish in the vessels, an increased growth in terms of total length and wet weight at test end was observed with increasing concentrations. This effect was statistically significantly different to controls at 33.3 µg/L and 76.8 µg/L (mean measured concentration; Williams t-test, two-sided, p<0.05), depending on the different measures and dependent on the sex of the fish. As this effect was clearly associated with the lower density of the fish in the vessels and as increased growth is not considered negative for fish, this effect was not considered to be primarily test-item related but rather as a beneficial effect on growth due to the reduction of post-hatch survival.

Regarding the endocrine-related endpoints determined during the FSDT, it was observed that triphenyl phosphate did not lead to an effect on the sex ratio. A histopathological analysis of fish gonads revealed that with increasing test item concentrations, the gonads reached a more mature stage. The maturation stage was determined for the test level presenting the NOEC of the endpoint post-hatch survival (3.01 µg triphenyl phosphate/L) and the highest test level (76.8 µg triphenyl phosphate/L). This effect was statistically significant for the testis at the highest treatment level (Students t-test, two-sided, p<0.05). For ovaries, this effect was not statistically significant. Additionally, to the maturation stages, further histopathological parameters were examined for these two test levels. No statistically significant differences were observed for the pathologies egg debris, increased oocyte atresia, granulomatomous inflammation and testis-ova.
Additionally, to the histopathological evaluations, the biomarker plasma vitellogenin (VTG) was measured in males and females. For VTG, no statistically significant differences to controls were observed at any treatment level in both sexes. Furthermore, the plasma levels of the steroid 17-beta estradiol (E2) were determined in females, and those of 11-keto testosterone (11-kT) in males. An increase in steroid concentrations was observed, with a statistically significant difference in E2 levels in females at a concentration of 76.8 µg triphenyl phosphate/L (mean mesured, Williams t-test, two-sided, p<0.05). For the 11-keto testosterone no statistically significant difference between controls and treatments were found. Thus, the NOEC for the concentration of the steroid 11-kT in male blood plasma was determined to be ≥ 76.8 µg triphenyl phosphate/L (mean measured).
The effects on gonad maturation as well as on increased steroid levels can also be considered to be related to the reduced number of fish in the test vessels with increasing concentrations, resulting in a supported growth and faster maturation of the fish. This results in a facilitated maturation of the gonads, accompanied by elevated steroid levels.
Thus, no endocrine-related effect of the test item could be determined.

Furthermore, effects on the liver and heart were determined. For liver, all test concentrations were examined, while for heart, additional groups were set (two replicates each of the control and the highest test level) in order to rule out any effect on this organ.
No statistically significant effect on the severity of the different lesions was determined.
For lesions of the heart, no statistics could be performed due to the limited number of replicates and test concentrations. However, effects were mainly observed in treatment conditions, however in a limited number of fish and always at mild severity. Thus, a strong effect of the test item on heart pathology was not evident.